A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon.

www.ajhg.org The American Journal of Human Genetics Volume 79 September 2006 439
ARTICLE
A Chromosome 8 Gene-Cluster Polymorphism with Low Human
Beta-Defensin 2 Gene Copy Number Predisposes to Crohn
Disease of the Colon
Klaus Fellermann, Daniel E. Stange, Elke Schaeffeler, Hartmut Schmalzl, Jan Wehkamp,
Charles L. Bevins, Walter Reinisch, Alexander Teml, Matthias Schwab, Peter Lichter,
Bernhard Radlwimmer, and Eduard F. Stange
Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has
been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The
DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy
population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin–
gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based
comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2
(HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range
2–10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with
inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4,
whereas those with colonic CD had a median of only 3 copies per genome ( for the surgical cohort;Pp .008 Pp .032
for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared
with controls ( for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals withPp .002
�3 copies have a significantly higher risk of developing colonic CD than did individuals with �4 copies (odds ratio 3.06;
95% confidence interval 1.46–6.45). An HBD-2 gene copy number of !4 was associated with diminished mucosal HBD-
2 mRNA expression ( ). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposesPp .033
to colonic CD, most likely through diminished beta-defensin expression.
From the Department of Internal Medicine I, Robert-Bosch-Hospital (K.F.; H.S.; J.W.; E.F.S.), and Dr. Margarete Fischer-Bosch-Institute for Clinical
Pharmacology, (E.S.; M.S.) Stuttgart; Division of Molecular Genetics, German Cancer Research Center, Heidelberg (D.E.S.; P.L.; B.R.); Department of
Medical Microbiology and Immunology, University of California–Davis, Davis (J.W.; C.L.B.); and Department of Internal Medicine IV, Medical University
of Vienna, Vienna (W.R.; A.T.)
Received February 20, 2006; accepted for publication May 12, 2006; electronically published July 12, 2006.
Address for correspondence and reprints: Dr. Klaus Fellermann, Robert-Bosch-Hospital, Auerbachstrasse 110, 70376 Stuttgart, Germany. E-mail: klaus
.fellermann@rbk.de
Am. J. Hum. Genet. 2006;79:439–448. � 2006 by The American Society of Human Genetics. All rights reserved. 0002-9297/2006/7903-0007$15.00
Crohn disease (CD [MIM 266600]) is a severe chronic in-
flammatory bowel disease characterized by intestinal ul-
ceration that affects predominantly the ileum and co-
lon.1,2 The cause of the disease is unknown. Recent find-
ings have suggested that the mucosal immunological re-
action is directed against the resident bacterial flora rather
than against tissue antigens.3 This loss of tolerance to the
normal flora may be due to a dysregulation of the gut
mucosal immune response4 or, alternatively, a break in the
antibacterial barrier where microbiota can trigger a dele-
terious immune response.5
CD appears to be a consequence of both genetic and
environmental influences. The 50% concordance rate in
MZ twins, who often exhibit the same phenotype,6 sug-
gests a rather balanced impact of genetic and such envi-
ronmental factors as smoking7,8 or childhood hygiene.9
During a genomewide search, several susceptibility loci
and genes—including NOD2 (CARD15) (MIM 605956),10,11
DLG5 (MIM 604090),12 and OCTN (MIM 604190 and
603377)13—have been found to be associated with CD.
The best replicated is NOD2, which is involved in intra-
cellular sensing of bacterial muramyl dipeptide, promi-
nently expressed in macrophages and in particular small
intestinal Paneth cells.14,15 Despite these significant ad-
vances, the multiple susceptibility loci and other genetic
factors hitherto identified16 do not satisfactorily explain
the inheritance rates.
The clinical syndrome of CD is variable with respect to
age at diagnosis, location (small and/or large intestine),
and disease behavior (inflammatory, stricturing, or pene-
trating disease). Therefore, these behavior parameters were
used in the Vienna classification,17 recently modified in
Montreal.18 Location of disease involvement proved to be
stable over time in individual patients, although the bio-
logical basis of small- versus large-intestinal involvement
is unclear. NOD2 mutations have been shown to be pre-
dominantly associated with ileal disease and have been
reported to be related to a relative lack of ileal Paneth-cell
alpha-defensins HD-5 (MIM 600472) and HD-6 (MIM
600471).19 Defensins are endogenous antibiotic peptides
that form a chemical barrier at the epithelial surface, and
their relative deficiency may lead to bacterial adherence
to the mucosa, slow invasion, and secondary mucosal
inflammation.5,20

440 The American Journal of Human Genetics Volume 79 September 2006 www.ajhg.org
Table 1. Number of Patients in the Cohorts
Examined for HBD-2 Gene Copy Number
Cohort
No. of Subjects
Cleveland Stuttgart Vienna
Control 20 149
CD: 85 54 111
Ileal (L1) 60 22 38
Colonic (L2) 25 10 36
Ileal and colonic (L3) 22 37
UC 38 37
In contrast to ileal disease, colonic CD is characterized
by an impaired induction of the epithelial beta-defensins
HBD-2 (MIM 602215), HBD-3 (MIM 606611), and HBD-
4.21–23 This relative deficiency of several beta-defensins is
unlikely to be explained by multiple coincident mu-
tations or other genetic alterations of all these genes.
Detailed studies of the defensin locus on chromosome
8 have uncovered an extensive DNA copy number poly-
morphism (CNP) of a gene cluster, including, among
others, the human beta-defensin genes HBD-2 (DEFB4),
HBD-3 (DEFB103), and HBD-4 (DEFB104).24 It has been
shown that the number of gene copies is positively cor-
related with the expression of HBD-2 in leukocytes.24 Since
all these defensins were found to be coordinately under-
expressed in colonic CD,21 we hypothesized that this par-
ticular phenotype may be associated with a low beta-de-
fensin gene cluster copy number. We therefore measured
DNA copy number in the beta-defensin cluster in two in-
dependent cohorts with inflammatory bowel diseases and
related it to mucosal HBD-2 gene expression.
Material and Methods
Patients
The patients from three separate cohorts received diagnoses, with
use of the same standard criteria, in Cleveland, Stuttgart, and
Vienna and were treated in specialized tertiary-care out- and in-
patient centers. Patients gave their informed consent, and local
ethics committees approved the study protocols. A small cohort—
of patients with colonic CD ( ) and healthy control indi-np 10
viduals ( )—was recruited in Stuttgart. Their blood-derivednp 10
genomic DNA was subjected to the microarray analysis described
below. The exploratory cohort was a surgical group of patients
who underwent surgical resection at the Cleveland Clinic for
treatment of ileal or colonic Crohn disease (table 1). The confir-
matory cohort were all whites with CD or ulcerative colitis (UC
[MIM 191930]) who were treated at the Robert-Bosch-Hospital
(Stuttgart) or the University Hospital (Vienna) (table 1). Diagnos-
tic and (Vienna) classification criteria were the same at both Eu-
ropean centers.17 In this classification, L1 is defined as ileal disease
only, L2 as colonic disease only, and L3 as ileal as well as colonic
disease. Finally, the Stuttgart control group was combined with
blood donors ( ), individuals unaffected by inflammatorynp 103
bowel disease who underwent surveillance colonoscopy (np
), and 20 control individuals unaffected by intestinal disease46
from Cleveland. All colonoscopies that included biopsies were
performed on patients from the Stuttgart cohort.
Array CGH (Array-Based Comparative Genomic
Hybridization) Analysis
DNA preparation, labeling, hybridization, and analysis procedures
were performed as published elsewhere.25,26 In brief, genomic DNA
from fresh-frozen blood obtained from 10 patients with colonic
CD and from 10 healthy control individuals was isolated using
the Blood and Cell Culture Kit (Qiagen) following the instructions
of the supplier. Sample DNA and reference DNA (pooled DNA
from six healthy individuals) were labeled differentially with use
of the Bioprime Labelling Kit (Invitrogen) and were hybridized
on a DNA microarray consisting of ∼8,000 genomic fragments
covering the human genome at a resolution of ∼0.5 Mb.27 For
the beta-defensin locus at 8p23.1, all additional genomic frag-
ments that were available at the time were added to the micro-
array to enhance the resolution in the region of interest. The
chromosomal mapping information was based on the Ensembl
Genome Browser release 36.35i (December 2005). Arrays were
scanned, and fluorescence intensities of all spots were filtered
(intensity/local background 13; mean/median intensity !1.3; SD
of genomic fragment log ratios !0.25) and were normalized block-
wise. Chromosomal breakpoints delimiting regions of different
copy number status were detected by GLAD (gain and loss anal-
ysis of DNA).28
Determination of HBD-2 Gene Copy Number
A TaqMan real-time PCR assay, specifically for amplification of
genomic HBD-2, was established by using a specific set of am-
plification primers (forward 5′-CACCTGTGGTCTCCCTGGAA-3′;
reverse 5′-AGCTTCTTGGCCTCCTCATG-3′) and a probe (6-FAM-
ATGCTGCAAAAAG-MGB). Quantitative HBD-2 amplification data
were normalized to ALB (albumin [MIM 103600]) as an internal
reference gene, which was coamplified simultaneously in a single-
tube biplex assay. The primers and probe for HBD-2 were designed
using Primer Express software, version 1.5 (Applied Biosystems).
For albumin, we used the primers and probe that were published
elsewhere.29 Primers were purchased from MWG-Biotech, and
probes were obtained from Applied Biosystems. Real-time PCR
was performed using the ABI Prism 7700 sequence-detection sys-
tem. Amplification reactions (25 ml each) were performed in trip-
licate with 20 ng of template DNA, 1# TaqMan Universal Master
Mix buffer (Applied Biosystems), 300 nM of each primer, and 200
nM of each fluorogenic probe. Thermal cycling was initiated with
a 2-min incubation at 50�C, followed by a first denaturation step
of 10 min at 95�C and then by 40 cycles for 15 s at 95�C and for
1 min at 60�C. In each assay, a standard curve was recorded and
a no-template control was included. To amplify HBD-2 and al-
bumin in a one-tube biplex assay, limiting primer conditions were
identified, to avoid competition of the two reactions. Quantifi-
cation was performed by both the standard-curve method and
the comparative CT (threshold cycle) method, as described else-
where.29 The assay was validated with a selection of DNA samples,
genotyped elsewhere (kindly provided by E. J. Hollox, Notting-
ham, United Kingdom), that contained 3, 4, 5, and 7 HBD-2 gene
copies.24
Real-Time Quantitative PCR
Frozen biopsies were disrupted in 1 ml of Trizol (Gibco BRL) until
complete fragmentation occurred. Total RNA was extracted ac-
cording to the supplier’s protocol. RNA quality was determined

www.ajhg.org The American Journal of Human Genetics Volume 79 September 2006 441
by electrophoresis and was quantified by photometry. Subse-
quently, 2 mg of RNA was reverse transcribed with oligo-dT prim-
ers and 200 U Superscript (Gibco BRL), according to the routine
procedure.
cDNA samples were subjected to real-time PCR as outlined else-
where.21 In brief, an aliquot corresponding to 50 ng of RNA was
set up in a 20-ml reaction mixture containing 4 mM MgCl2, 0.5
mM of each primer (for HBD-2, forward 5′-ATCAGCCATGAGG-
GTCTTGT-3′ and reverse 5′-GAGACCACAGGTGCCAATTT-3′ [an-
nealing temperature 60�C; product 172 bp]; for HBD-3, forward
5′-TGAAGCCTAGCAGCTATGAGGATC-3′ and reverse 5′- CCGCC-
TCTGACTCTGCAATAA-3′ [annealing temperature 62�C; product
128 bp]; for interleukin 8 [IL-8 (MIM 146930)], forward 5′-ATGACT-
TCCAAGCTGGCCGTGGC-3′ and reverse 5′-TCTCAGCCCTCTT-
CAAAAACTTC-3′ [annealing temperature touch down protocol
66�C–60�C; product 292 bp] [Sigma]) and 1# LightCycler-Fast-
Start DNA Master SYBR Green I Mix (Roche Molecular Biochemi-
cals) and was loaded in capillary columns. PCR was performed
for 45 cycles in a LightCycler (Roche Molecular Biochemicals).
After each cycle, fluorescence emission readings reflecting the
increase in PCR products were monitored and analyzed using
LightCycler software (Roche Molecular Biochemicals).
DNA Sequencing
Exons 1 and 2 of the HBD2 gene and ∼50 bp of adjacent non-
coding regions were PCR amplified from genomic DNA and were
sequenced on an Applied Biosystems 3100 capillary sequencer
with use of Big-Dye chemistry. The sequences of the amplification
and nested sequencing primers are available on request.
Statistics
Statistical comparisons of copy numbers of (1) ileal and colonic
subsets in the Cleveland cohort as well as (2) patients with L1,
L2, and L3 UC and (3) control individuals from the European
collective were performed with the (two-sided) Mann-Whitney
test. Additionally, Kruskal-Wallis analysis of variance of ranks,
including post hoc assessment by Dunn’s test, was performed to
correct for multiple testing. Differences in copy number distri-
bution among the clinical cohorts were assessed by the Pearson
x2 test, with continuity corrections. Again, the Mann-Whitney
test was used to assess differences in HBD-2 mRNA expression
with increasing HBD-2 gene copy numbers.
Results
Microarray Analysis
Using an array CGH with ∼8,000 genomic fragments cov-
ering the human genome, with an average resolution of
∼0.5 Mb, we screened 10 patients with colonic CD and
10 healthy control individuals for DNA copy number var-
iations. No gross chromosomal aberrations were present
in either group (data not shown), but we detected several
known regions of CNP, such as the IGHG1 gene cluster at
14q32.33 (MIM 147100) (data not shown) or the amylase
gene cluster at 1p21.1 (MIM 104700) (fig. 1A). Except for
the beta-defensin gene cluster at 8p23.1 (fig. 1B), however,
no region showed a bias toward copy number loss or gain
in patients versus controls. In the Ensembl Genome Browser
(release 36.35i), the beta-defensin cluster is shown to be
organized as a pair of inverted repeats separated by a se-
quence gap (fig. 1C). This probably represents the minimal
size of the locus in humans. In the patient and control
samples that showed copy number variation compared
with the control DNA, the size of the variable region was
always the same, covering ∼900 kb (megabase 7.1–8.0),
including BAC clones RP11-278P18, RP11-1005B13, and
RP11-52B19 (Clone Registry). This region contains both
copies of the beta-defensin cluster but not the alpha-de-
fensin cluster, as is shown for patient X1604 in figure 1C.
When comparing the copy number profiles of the patient
and control groups, it became evident that patients with
CD, on average, seemed to have fewer copies than did
healthy individuals. Relative to a DNA pool from 6 healthy
control individuals, 8 of 10 patients with CD displayed
small copy number losses, and none displayed copy num-
ber gains (fig. 1D). In control individuals, no such bias
was observed (fig. 1E). The neighboring alpha-defensingene
cluster (fig. 1C), which mapped to megabase 6.76–6.90, and
other beta-defensin–like loci—identified on chromosomal
bands 6p12, 20q11.1, and 20p13 by sequence similarity
search30—did not show any copy number variation in pa-
tients or control individuals (data not shown).
HBD-2 Gene Copy Numbers in Inflammatory Bowel
Diseases
Our array CGH analyses clearly indicated that low DNA
copy number at the main beta-defensin locus might be
connected to CD; however, the size of this initial sample
was much too small for reliable correlation analysis. Fur-
thermore, whereas array CGH is extremely useful for whole-
genome scans, its quantitative performance is suboptimal
at loci that are rich in low copy number DNA repeats
(LCRs), such as the beta-defensin cluster. At such loci, the
reported copy number ratios can be severely affected by
cross-hybridization of the LCRs.25
Therefore, we decided, as an alternative, to use quanti-
tative PCR analysis of the HBD-2 gene to estimate the DNA
copy number of the beta-defensin cluster. This HBD2
gene–specific approach was applied to a control popula-
tion and to two patient cohorts, one from the United
States and one from Europe (table 1). In the control pop-
ulation ( ) the copy numbers had a range of 2–10np 169
per genome, with a median number of 4 copies (fig. 2A).
The numbers of control individuals who carry the median
(4), below-median (!4), or above-median (14) number of
copies were about equal. Details of the copy number fre-
quencies of all cohorts and subgroups are given in table
2.
The U.S. patient cohort from the Cleveland Clinic con-
sisted of 85 surgical patients with CD who had indications
for ileal versus colonic resection. In patients with ileal re-
sections (fig. 2B), the median number of copies was iden-
tical to that of the control group (4 copies); also, the fre-
quency distribution of the three subgroups (with !4, with
4, and with 14 gene copy numbers) was not significantly

442 The American Journal of Human Genetics Volume 79 September 2006 www.ajhg.org
Figure 1. A, Copy number ratios of ∼8,000 clones covering the whole genome, with a resolution of ∼0.5 Mb, are shown for patient
X1604. The loci of the amylase and beta-defensin gene clusters are indicated. The red lines in panels A and B show the smoothed copy
number ratio, as calculated by the GLAD algorithm. B, Copy number ratios of all clones on chromosome 8 of patient X1604. The beta-
defensin locus on 8p23.1 is indicated. C, Detail of copy number ratios on 8p23.1 for patient X1604. The alpha-defensin and the two
beta-defensin loci are shown by arrows, indicating the genomic orientation of the loci; “sequence gap” indicates a region where no
human reference sequence is available. The genomic position, length, and name of genomic fragments covering the region of interest
are displayed at the top. The red line indicates the average genomic position of the region that shows a CNP in our 20 hybridizations.
D, Copy number ratios in the 8p23.1 region of 10 patients with colonic CD. E, Copy number ratios in the 8p23.1 region of 10 healthy
control individuals.
different from that of the control group. In contrast, the
majority (72%) of patients with colonic resections had a
copy number !4, with a median of 3 copies (fig. 2C). The
difference in copy numbers was highly significant (Pp
[Mann-Whitney]) between the ileal and colonic sub-.008
groups with CD. This was maintained when tested by
Kruskal-Wallis analysis of variance ( for both postP ! .01
hoc Dunn test ileal vs. colonic and colonic vs. control).
Similarly, the proportion of the three copy number groups
in the two subgroups with CD was significantly different
( [Pearson x2]).Pp .018
An independent second cohort of European patients with
CD ( ) from Stuttgart and Vienna was classified ac-np 165
cording to the Vienna classification of location into those
with ileal disease only (L1), with colonic disease only (L2),
or with ileal plus colonic disease (L3). Again, ileal CD (L1)
exhibited a copy number distribution similar to controls
( ), with a majority in the group with 4 gene copiesP 1 .05
(fig. 3). The median was 4 copies in L1 and controls com-
pared with 3 in L2 ( and , respectivelyPp .032 Pp .001
[Mann-Whitney]). Analysis of variance and post hoc test
identified a significant difference between L2 and controls

www.ajhg.org The American Journal of Human Genetics Volume 79 September 2006 443
Figure 2. Distribution of HBD-2 gene copy numbers in the 169
controls from Stuttgart and Cleveland (A) and both surgical CD
cohorts with ileal (B) and colonic resection (C) allocated to !4,
4, or 14 copies per genome. The difference in distribution between
ileal and colonic resection was significant ( [x2 test]).Pp .018
Table 2. HBD-2 Gene Copy Number Frequencies
Cohort
Percentage of Cohort with
HBD-2 Gene Copy Number of
2 3 4 5 6 7 8 9 10
Control:
Cleveland 5.0 15.0 45.0 25.0 5.0 5.0
Europe 2.7 23.5 38.3 23.5 10.1 .7 .7 .7
Cleveland:
Ileal 1.7 36.7 35.0 20.0 6.7
Colonic 12.0 60.0 16.0 8.0 4.0
European:
L1 5.0 23.3 43.3 21.7 1.7 5.0
L2 2.2 50.0 32.6 10.9 2.2 2.2
L3 8.5 32.2 35.6 15.3 5.1 3.4
UC 5.3 33.3 33.3 14.7 9.3 2.7 1.3
NOTE.—For the number of patients in these subgroups, see table 1.
only ( ). Colonic CD only (L2) was characterized byP ! .05
a shift to lower copy numbers, with the majority (52%)
carrying !4 copies of HBD-2 ( vs. controls;Pp .002 Pp
vs. L1 [Pearson x2]). Individuals with �3 copies have.037
a significantly higher risk of developing colonic CD than
do individuals with �4 copies (odds ratio 3.06; 95% CI
1.46–6.45). Patients with L3 showed an intermediate dis-
tribution pattern between controls and L2, and, although
the median was the normal 4 copies, the shift in distri-
bution was significant ( [Mann-Whitney]). In UC,Pp .034
differences with controls were not significant. Detailed
noncategorized copy number data are given in table 2.
HBD-2 Expression Related to HBD-2 Gene Copy Numbers
in Inflamed Mucosa
Mucosal biopsies were analyzed for HBD-2, HBD-3, and
IL-8 expression in patients from the Stuttgart cohort. Since
HBD-2 expression is negligible in normal mucosa but is
enhanced during inflammation, the biopsy samples were
taken from a subgroup of 44 patients with inflammation
due to CD ( ) or UC ( ). HBD-2 and HBD-3np 17 np 27
expression was highly correlated ( in CD;rp 0.84 rp
in UC). When the mucosal mRNA expression of HBD-0.86
2 was related to the HBD-2 gene copy number in the same
patients (fig. 4), HBD-2 mRNA expression was significantly
diminished in the group with !4 compared with 4 copies
( [Mann-Whitney]) or �4 ( ), whereas IL-Pp .023 Pp .033
8 expression in these copy number groups was not sig-
nificantly different (data not shown).
HBD-2 Gene Sequencing
Finally, to investigate the possibility that HBD-2 gene ex-
pression might be affected by the presence of gene copies
that are inactivated by point mutations, we sequenced
exon 1 and exon 2 of the HBD-2 gene in eight patients
with colonic CD and eight control individuals. We did not
find any nonsense or missense mutations in the coding
region. The only sequence variations present were one
synonymous SNP in exon 2 (rs2740090) and four SNPs in
noncoding sequences (rs2740086, rs2740091, rs2737912,
and rs2737913). These SNPs occurred without predomi-
nance in either patients or control individuals (data not
shown; SNPs were retrieved from dbSNP).
Discussion
CNPs and their effect on phenotypic variation and disease
recently have become a major focus of attention.31–33 At
present, 1200 loci of large-scale CNPs have been detected

444 The American Journal of Human Genetics Volume 79 September 2006 www.ajhg.org
Figure 3. Distribution of HBD-2 gene copy numbers (!4, 4, or 14 copies per genome) in the European cohort as categorized into
ileal disease (L1) (A), colonic disease (L2) (B), ileal and colonic disease (L3) (C), and UC (D). L2 (colonic CD) differed significantly
from L1 (ileal CD) and controls ( and , respectively [x2 test]).Pp .037 Pp .002
using BAC arrays (Clone Registry)32,33 or oligonucleotide
arrays.31 With use of SNP genotyping data, recent studies
have found ∼1,000 fine-scale deletion variants in the hu-
man genome.34–36 The role of these genetic alterations and
their impact on disease or disease susceptibility is not clear.
Several examples show that copy number alterations can
lead to altered gene expression and disease; for example,
copy number increase at the alpha-synuclein locus (4q21
[MIM 163890]) causes Parkinson disease (MIM 168600),37
and duplication of PMP22 (17q11.2 [MIM 601097]) causes
Charcot-Marie-Tooth disease (MIM 118220).38 Recently, a
study showed that some cases of early-onset Alzheimer
disease (MIM 104300) resulted from a duplication of the
APP locus (MIM 104760).39 CNPs can also lead to disease
susceptibility, which was shown, for example, for the pos-
session of a low copy number of the CCL3L1 gene (MIM
601395) that is associated with markedly enhanced HIV/
AIDS (MIM 609423) susceptibility.40 Gene CNPs, which are
associated with variable drug response, have also been de-
scribed for drug-metabolizing enzymes. For example, in-
herited amplification of an active gene in the cytochrome
P450 CYP2D locus is a cause of ultrarapid metabolism of
debrisoquine.41 We have recently reported the development
of an RT-PCR–based assay to genotype CYP2D6 (MIM
124030) with respect to its gene copy number,29 similar
to the technique used in the present study. A previous
study of beta-defensin gene copy number in cystic fibrosis
(MIM 219700) had negative results.42
Recently, the finished sequence and a gene catalogue of
chromosome 8 were reported.43 A unique feature of the
chromosome is a vast region of ∼15 Mb on distal 8p that
appears to have a strikingly high mutation rate. Within
this region, the CNPs in the defensin cluster on chromo-
some 8p23.1 are of special interest, since they may be
linked to immune function and disease. The copy number
variation in this region is complex, with two different
clusters of defensins—alpha- and beta-defensins—appar-
ently showing an independent variation of their copy
numbers.44 In the alpha-defensin cluster, the genes DEFA1
(MIM 125220) and DEFA3 (MIM 604522) code for the neu-
trophil defensins HNP-1 and HNP-3, respectively. Their
copy numbers, with a range of 5–14, were related to neu-
trophil HNP 1–3 levels.44 In contrast, copy numbers of
DEFA5 (MIM 600472) and DEFA6 (MIM 600471)—coding
for the alpha-defensins HD-5 and HD-6, respectively—ap-
pear to be stable, with two per diploid genome.44 This was
also confirmed for the patients with CD, which suggests
that the low HD-5 and HD-6 levels in ileal CD are not
related to low copy numbers.45
Prompted by the diminished induction of beta-defen-
sins in the colonic mucosa of patients with CD compared
with patients with UC,21–23 we asked whether the copy
numbers in the variable beta-defensin locus were low with
respect to those in both healthy control individuals and
subjects with inflammatory bowel disease (UC and ileal
CD). The initial experiments, with use of high-resolution
array CGH for genomic copy number profiling, suggested
that, compared with controls. there indeed was a lower
copy number of the beta-defensin locus in patients with
colonic CD. All other regions of CNPs covered by the mi-
croarray showed equal copy number distributions between
patients with colonic CD and control individuals. A se-

www.ajhg.org The American Journal of Human Genetics Volume 79 September 2006 445
Figure 4. HBD-2 mRNA expression, with respect to HBD-2 gene
copy number, in mucosal specimens from patients with CD and UC
with rectal inflammation.
quencing approach of the HBD-2 gene in a limited number
of patients with colonic CD and control individuals re-
vealed several known SNPs but no nonsense or missense
mutations. None of the SNPs was preferentially found in
either patients or control individuals. Further haplotype
analysis of the HBD-2 gene was not performed because of
the immense difficulties of such analyses.46
It might seem surprising that the beta-defensin locus on
chromosome 8 has not been identified in any of the pre-
vious linkage studies,10,11 in particular since deletions re-
cently were shown to be in linkage disequilibrium with
SNPs.35 The relevance of this finding for the present study
is questionable, however, since the beta-defensin cluster
consists of multiple gene copies and is expected to be
much less stable than a simple deletion. Therefore, it is
not clear whether gene CNPs are at all detectable by link-
age analyses. Furthermore, previous studies might have
overlooked a possible linkage, since they did not distin-
guish colonic and ileal CD.
Since DNA microarrays allow full coverage of the human
genome but are less suitable for high-throughput studies
in large patient cohorts, we developed a real-time PCR-
based technique to quantitate HBD-2 copy number vari-
ations. Since HBD-2 and HBD-3 copy numbers covary in
tandem, it may be assumed that the measured HBD-2 copy
number reflects the whole beta-defensin gene cluster.24,44
This is also consistent with the close correlation of HBD-
2 and HBD-3 expression demonstrated in the present and
a previous study.21 A similar PCR-based technique was re-
cently published by Chen et al.47 The normal median num-
ber of 4 copies found in the present study confirms the
finding of Chen et al.47 and that of Hollox et al.42 but is
slightly lower than the median of 5 observed by Linzmeier
et al.44
Since the surgical patients with CD usually have a par-
ticularly severe disease course, we first studied patients with
ileal versus colonic resections as typical phenotypes. A
clear-cut difference by 1 gene copy was observed and was
then confirmed in a second, larger cohort of patients clas-
sified clinically according to the Vienna classification.17
We focused on localization as a stable factor during the
disease course and noted that the patient cohort with co-
lonic disease experienced a shift to lower copy numbers.
Although this shift was significant, there was clearly an
overlap between the groups, which suggests that addi-
tional, still unknown factors of genetic or environmental
origin impact CD. Interestingly, patients with both ileal
and colonic CD (L3) had an intermediate copy number
distribution.
Notably, this relatively small difference in HBD-2 copy
numbers appeared to have an influence on mucosal HBD-
2 expression by gene dosage effect,48 which was signifi-
cantly higher in patients with 4 compared with those with
fewer gene copies. The biological consequence will likely
be an impaired antimicrobial defense in individuals with
!4 copies. This is consistent with the previous finding of
Hollox et al.24 that HBD-2 expression in lymphoblastoid
cell lines was correlated linearly with the HBD-2 gene copy
number in the range of 2–7 copies. In some contrast, we
did not observe a linear increase in the higher range of
copy numbers 14, which may be related to the present
complex patient situation in which many other factors,
including bacterial flora and inflammation, may affect de-
fensin expression. Alternatively, the plateau in defensin
gene expression we observe at copy numbers of �4 might
be due to some unknown mechanism limiting gene ex-
pression in the native colon tissue, which might not be
active in lymphoblastoid cells. At any rate, despite these
confounding factors, the gene dosage effect on HBD-2 ex-
pression prevailed for low-to-normal copy numbers.
In conclusion, the low beta-defensin gene copy number
in CD was clearly associated with colonic disease localiza-
tion—that is, a specific phenotype of CD. A normal beta-
defensin copy number distribution was seen in CD of the
ileum and also in UC. This study may provide the genetic
basis for the diminished induction of beta-defensins in
colonic CD compared with UC in which colonic beta-
defensin induction is intact, as described elsewhere.21–23
We suggest that the antibacterial barrier in the colonic
mucosa is weakened because of relative defensin defi-
ciency in colonic CD. This finding complements our pre-
vious report of a diminished alpha-defensin synthesis in
ileal CD, which was further pronounced with a concom-
itant NOD2 mutation.19,45 This differential compromise of
the complex defensin system may explain the different
phenotypes of colonic versus ileal CD20,49 as well as the
observation that bacteria of the normal flora adhere and
sometimes invade the epithelium in CD.50–52 Possibly, this
barrier problem triggers the chronic inflammation known
as CD.

446 The American Journal of Human Genetics Volume 79 September 2006 www.ajhg.org
Acknowledgments
This study was supported by the Robert Bosch Foundation, by
National Institutes of Health grant AI32738 (to C.L.B.), by the
Deutsche Forschungsgemeinschaft Graduiertenkolleg 886 (to
D.E.S.), and by the Bundesministerium fu¨r Bildung und For-
schung nationales Genomforschungsnetzwerk 01GS0460 (to
P.L.) and 01GR0417 (to B.R.). We thank Falk Schubert (Division
of Theoretical Bioinformatics, German Cancer Research Center,
Heidelberg), for excellent support with the statistical analyses,
and Daniel Mertens, for assistance with sequence analysis. We
also thank Drs. Victor Fazio, Bo Shen, and others of the Inflam-
matory Bowel Disease Center (The Cleveland Clinic Foundation),
and we thank the colleagues at the Robert-Bosch-Hospital (Stutt-
gart) for help with sample procurement. Last but not least, we
are indebted to the excellent work of our technicians.
Web Resources
The URLs for data presented herein are as follows:
Clone Registry, http://www.ncbi.nlm.nih.gov/genome/clone/
Ensembl Genome Browser, http://dec2005.archive.ensembl.org/
(for release 36.35i [December 2005])
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi
.nlm.nih.gov/Omim/ (for CD, NOD2 [CARD15], DLG5, OCTN,
HD-5, HD-6, HBD-2, HBD-3, HBD-4, UC, albumin, IL-8, IGHG1,
amylase, alpha-synuclein, Parkinson disease, PMP22, Charcot-
Marie-Tooth disease, early-onset Alzheimer disease, APP, CCL3L1,
HIV/AIDS, CYP2D6, cystic fibrosis, DEFA1, DEFA3, DEFA5, and
DEFA6)
dbSNP, http://www.ncbi.nlm.nih.gov/SNP/
References
1. Loftus EV Jr (2004) Clinical epidemiology of inflammatory
bowel disease: incidence, prevalence, and environmental in-
fluences. Gastroenterology 126:1504–1517
2. Shanahan F (2002) Crohn’s disease. Lancet 359:62–69
3. Duchmann R, Kaiser I, Hermann E, Mayet W, Ewe K, Meyer
zum Bu¨schenfelde K-H (1995) Tolerance exists towards resi-
dent intestinal flora but is broken in active inflammatory
bowel disease. Clin Exp Immunol 102:448–455
4. Duchmann R, May E, Heike M, Knolle P, Neurath M, Meyer
zum Bu¨schenfelde K-H (1999) T cell specificity and cross reac-
tivity towards enterobacteria, Bacteroides, Bifidobacterium,and
antigens from resident intestinal flora in humans. Gut 44:
812–818
5. Fellermann K, Wehkamp J, Herrlinger KR, Stange EF (2003)
Crohn’s disease: a defensin deficiency syndrome? Eur J Gas-
troenterol Hepatol 15:627–634
6. Halfvarson J, Bodin L, Tysk C, Lindberg E, Jarnerot G (2003)
Inflammatory bowel disease in a Swedish twin cohort: a long-
term follow-up of concordance and clinical characteristics.
Gastroenterology 124:1767–1773
7. Tobin MV, Logan RF, Langman MJ, McConnell RB, Gilmore
IT (1987) Cigarette smoking and inflammatory bowel disease.
Gastroenterology 93:316–321
8. Bridger S, Lee JC, Bjarnason I, Jones JE, Macpherson AJ (2002)
In siblings with similar genetic susceptibility for inflamma-
tory bowel disease, smokers tend to develop Crohn’s disease
and non-smokers develop ulcerative colitis. Gut 51:21–25
9. Gent AE, Hellier MD, Grace RH, Swarbrick ET, Coggon D (1994)
Inflammatory bowel disease and domestic hygiene in infan-
cy. Lancet 343:766–767
10. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Be-
laiche J, Almer S, Tysk C, O’Morain CA, Gassull M, Binder V,
Finkel Y, Cortot A, Modigliani R, Laurent-Puig P, Gower-Rous-
seau C, Macry J, Colombel JF, Sahbatou M, Thomas G (2001)
Association of NOD2 leucine-rich repeat variants with sus-
ceptibility to Crohn’s disease. Nature 411:599–603
11. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos
R, Britton H, Moran T, Karaliuskas R, Duerr RH, Achkar JP,
Brant SR, Bayless TM, Kirschner BS, Hanauer SB, Nunez G,
Cho JH (2001) A frameshift mutation in NOD2 associated
with susceptibility to Crohn’s disease. Nature 411:603–606
12. Stoll M, Corneliussen B, Costello CM, Waetzig GH, Mellgard
B, Koch WA, Rosenstiel P, Albrecht M, Croucher PJ, Seegert
D, Nikolaus S, Hampe J, Lengauer T, Pierrou S, Foelsch UR,
Mathew CG, Lagerstrom-Fermer M, Schreiber S (2004) Ge-
netic variation in DLG5 is associated with inflammatory bowel
disease. Nat Genet 36:476–480
13. Peltekova VD, Wintle RF, Rubin LA, Amos CI, Huang Q, Gu
X, Newman B, Van Oene M, Cescon D, Greenberg G, Griffiths
AM, George-Hyslop PH, Siminovitch KA (2004) Functional
variants of OCTN cation transporter genes are associated with
Crohn disease. Nat Genet 36:471–475
14. Lala S, Ogura Y, Osborne C, Hor SY, Bromfield A, Davies S,
Ogunbiyi O, Nunez G, Keshav S (2003) Crohn’s disease and
the NOD2 gene: a role for Paneth cells. Gastroenterology 125:
47–57
15. Ogura Y, Lala S, Xin W, Smith E, Dowds TA, Chen FF, Zim-
mermann E, Tretiakova M, Cho JH, Hart J, Greenson JK, Kes-
hav S, Nunez G (2003) Expression of NOD2 in Paneth cells:
a possible link to Crohn’s ileitis. Gut 52:1591–1597
16. Vermeire S, Rutgeerts P (2005) Current status of genetics re-
search in inflammatory bowel disease. Genes Immun 6:637–
645
17. Gasche C, Scholmerich J, Brynskov J, D’Haens G, Hanauer
SB, Irvine EJ, Jewell DP, Rachmilewitz D, Sachar DB, Sandborn
WJ, Sutherland LR (2000) A simple classification of Crohn’s
disease: report of the Working Party for the World Congresses
of Gastroenterology, Vienna 1998. Inflamm Bowel Dis 6:8–15
18. Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN,
Brant SR, Caprilli R, Colombel JF, Gasche C, Geboes K, Jewell
DP, Karban A, Loftus EV Jr, Pena AS, Riddell RH, Sachar DB,
Schreiber S, Steinhart AH, Targan SR, Vermeire S, Warren BF
(2005) Toward an integrated clinical, molecular and serolog-
ical classification of inflammatory bowel disease: report of a
working party of the 2005 Montreal World Congress of Gas-
troenterology. Can J Gastroenterol Suppl A 19:5–36
19. Wehkamp J, Harder J, Weichenthal M, Schwab M, Schaffeler
E, Schlee M, Herrlinger KR, Stallmach A, Noack F, Fritz P, Schro-
der JM, Bevins CL, Fellermann K, Stange EF (2004) NOD2
(CARD15) mutations in Crohn’s disease are associated with
diminished mucosal a-defensin expression. Gut 53:1658–1664
20. Wehkamp J, Schmid M, Fellermann K, Stange EF (2005) De-
fensin deficiency, intestinal microbes, and the clinical phe-
notypes of Crohn’s disease. J Leukoc Biol 77:460–465
21. Wehkamp J, Harder J, Weichenthal M, Mu¨ller O, Herrlinger
KR, Fellermann K, Schro¨der JM, Stange EF (2003) Inducible
and constitutive b-defensins are differentially expressed in
Crohn’s disease and ulcerative colitis. Inflamm Bowel Dis 9:
215–223

www.ajhg.org The American Journal of Human Genetics Volume 79 September 2006 447
22. Fahlgren A, Hammarstrom S, Danielsson A, Hammarstrom
ML (2003) Increased expression of antimicrobial peptides and
lysozyme in colonic epithelial cells of patients with ulcerative
colitis. Clin Exp Immunol 131:90–101
23. Fahlgren A, Hammarstrom S, Danielsson A, Hammarstrom
ML (2004) b-Defensin-3 and -4 in intestinal epithelial cells
display increased mRNA expression in ulcerative colitis. Clin
Exp Immunol 137:379–385
24. Hollox EJ, Armour JAL, Barber JCK (2003) Extensive normal
copy number variation of a b-defensin antimicrobial-gene
cluster. Am J Hum Genet 73:591–600
25. Mendrzyk F, Korshunov A, Toedt G, Schwarz F, Korn B, Joos
S, Hochhaus A, Schoch C, Lichter P, Radlwimmer B (2006)
Isochromosome breakpoints on 17p in medulloblastoma are
flanked by different classes of DNA sequence repeats. Genes
Chromosomes Cancer 45:401–410
26. Stange DE, Radlwimmer B, Schubert F, Traub F, Pich A, Toedt
G, Mendrzyk F, Lehmann U, Eils R, Kreipe H, Lichter P (2006)
High-resolution genomic profiling reveals association of chro-
mosomal aberrations on 1q and 16p with histologic and ge-
netic subgroups of invasive breast cancer. Clin Cancer Res 12:
345–352
27. Mendrzyk F, Radlwimmer B, Joos S, Kokocinski F, Benner A,
Stange DE, Neben K, Fiegler H, Carter NP, Reifenberger G,
Korshunov A, Lichter P (2005) Genomic and protein expres-
sion profiling identifies CDK6 as novel independent prog-
nostic marker in medulloblastoma. J Clin Oncol 23:8853–
8862
28. Hupe P, Stransky N, Thiery JP, Radvanyi F, Barillot E (2004)
Analysis of array CGH data: from signal ratio to gain and loss
of DNA regions. Bioinformatics 20:3413–3422
29. Schaeffeler E, Schwab M, Eichelbaum M, Zanger UM (2003)
CYP2D6 genotyping strategy based on gene copy number
determination by TaqMan real-time PCR. Hum Mutat 22:476–
485
30. Schutte BC, Mitros JP, Bartlett JA, Walters JD, Jia HP, Welsh
MJ, Casavant TL, McCray PB Jr (2002) Discovery of five con-
served b-defensin gene clusters using a computational search
strategy. Proc Natl Acad Sci USA 99:2129–2133
31. Sebat J, Lakshmi B, Troge J, Alexander J, Young J, Lundin P,
Maner S, Massa H, Walker M, Chi M, Navin N, Lucito R, Healy
J, Hicks J, Ye K, Reiner A, Gilliam TC, Trask B, Patterson N,
Zetterberg A, Wigler M (2004) Large-scale copy number poly-
morphism in the human genome. Science 305:525–528
32. Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi
Y, Scherer SW, Lee C (2004) Detection of large-scale variation
in the human genome. Nat Genet 36:949–951
33. Sharp AJ, Locke DP, McGrath SD, Cheng Z, Bailey JA, Vallente
RU, Pertz LM, Clark RA, Schwartz S, Segraves R, Oseroff VV,
Albertson DG, Pinkel D, Eichler EE (2005) Segmental dupli-
cations and copy-number variation in the human genome.
Am J Hum Genet 77:78–88
34. McCaroll SA, Hadnott TN, Perry GH, Sabeti PC, Zody MC,
Barrett JC, Dallaire S, Gabriel SB, Lee C, Daly MJ, Altshuler
DM, International HapMap Consortium (2006) Common de-
letion polymorphisms in the human genome. Nat Genet 28:
86–92
35. Hinds DA, Kloek AP, Jen M, Chen X, Frazer KA (2006) Com-
mon deletions and SNPs are in linkage disequilibrium in the
human genome. Nat Genet 38:82–85
36. Conrad DF, Andrews TD, Carter NP, Hurles ME, Pritchard JK
(2006) A high-resolution survey of deletion polymorphisms
in the human genome. Nat Genet 38:75–81
37. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kach-
ergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, Lin-
coln S, Crawley A, Hanson M, Maraganore D, Adler C, Cook-
son MR, Muenter M, Baptista M, Miller D, Blancato J, Hardy
J, Gwinn-Hardy K (2003) a-Synuclein locus triplication causes
Parkinson’s disease. Science 302:841
38. Lupski JR, Oca-Luna RM, Slaugenhaupt S, Pentao L, Guzzetta
V, Trask BJ, Saucedo-Cardenas O, Barker DF, Killian JM, Garcia
CA, Chakravarti A, Patel PI (1991) DNA duplication associ-
ated with Charcot-Marie-Tooth disease type 1A. Cell 66:219–
232
39. Rovelet-Lecrux A, Hannequin D, Raux G, Le Meur N, La-
querriere A, Vital A, Dumanchin C, Feuillette S, Brice A, Ver-
celletto M, Dubas F, Frebourg T, Campion D (2006) APP locus
duplication causes autosomal dominant early-onset Alzhei-
mer disease with cerebral amyloid angiopathy. Nat Genet 38:
24–26
40. Gonzalez E, Kulkarni H, Bolivar H, Mangano A, Sanchez R,
Catano G, Nibbs RJ, Freedman BI, Quinones MP, Bamshad
MJ, Murthy KK, Rovin BH, Bradley W, Clark RA, Anderson
SA, O’Connell RJ, Agan BK, Ahuja SS, Bologna R, Sen L, Dolan
MJ, Ahuja SK (2005) The influence of CCL3L1 gene-contain-
ing segmental duplications on HIV-1/AIDS susceptibility. Sci-
ence 307:1434–1440
41. Johansson I, Lundqvist E, Bertilsson L, Dahl ML, Sjoqvist F,
Ingelman-Sundberg M (1993) Inherited amplification of an
active gene in the cytochrome P450 CYP2D locus as a cause
of ultrarapid metabolism of debrisoquine. Proc Natl Acad Sci
USA 90:11825–11829
42. Hollox EJ, Davies J, Griesenbach U, Burgess J, Alton EW, Ar-
mour JA (2005) Beta-defensin genomic copy number is not
a modifier locus for cystic fibrosis. J Negat Results Biomed 4:9
43. Nusbaum C, Mikkelsen TS, Zody MC, Asakawa S, Taudien S,
Garber M, Kodira CD, et al (2006) DNA sequence and analysis
of human chromosome 8. Nature 439:331–335
44. Linzmeier RM, Ganz T (2005) Human defensin gene copy
number polymorphisms: comprehensive analysis of inde-
pendent variation in a- and b-defensin regions at 8p22-p23.
Genomics 86:423–430
45. Wehkamp J, Salzman NH, Porter E, Nuding S, Weichenthal
M, Petras RE, Shen B, Schaeffeler E, Schwab M, Linzmeier R,
Feathers RW, Chu H, Lima H Jr, Fellermann K, Ganz T, Stange
EF, Bevins CL (2005) Reduced Paneth cell a-defensins in ileal
Crohn’s disease. Proc Natl Acad Sci USA 102:18129–18134
46. Taudien S, Galgoczy P, Huse K, Reichwald K, Schilhabel M,
Szafranski K, Shimizu A, Asakawa S, Frankish A, Loncarevic
IF, Shimizu N, Siddiqui R, Platzer M (2004) Polymorphic seg-
mental duplications at 8p23.1 challenge the determination
of individual defensin gene repertoires and the assembly of
a contiguous human reference sequence. BMC Genomics 5:92
47. Chen Q, Book M, Fang X, Hoeft A, Stuber F (2006) Screening
of copy number polymorphisms in human b-defensin genes
using modified real-time quantitative PCR. J Immunol Meth-
ods 308:231–240
48. Lupski JR (1999) Charcot-Marie-Tooth polyneuropathy: du-
plication, gene dosage, and genetic heterogeneity. Pediatr Res
45:159–165

448 The American Journal of Human Genetics Volume 79 September 2006 www.ajhg.org
49. Wehkamp J, Fellermann K, Herrlinger KR, Bevins CL, Stange
EF (2005) Mechanisms of disease: defensins in gastrointestinal
diseases. Nat Clin Pract Gastroenterol Hepatol 2:406–415
50. Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-
Baucke V, Ortner M, Weber J, Hoffmann U, Schreiber S, Dietel
M, Lochs H (2002) Mucosal flora in inflammatory bowel dis-
ease. Gastroenterology 122:44–54
51. Martin HM, Campbell BJ, Hart CA, Mpofu C, Nayar M, Singh
R, Englyst H, Williams HF, Rhodes JM (2004) Enhanced Esch-
erichia coli adherence and invasion in Crohn’s disease and
colon cancer. Gastroenterology 127:80–93
52. Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser
AL, Barnich N, Bringer MA, Swidsinski A, Beaugerie L, Col-
ombel JF (2004) High prevalence of adherent-invasive Esch-
erichia coli associated with ileal mucosa in Crohn’s disease.
Gastroenterology 127:412–421