A Chromosome 8 Gene-Cluster Polymorphism with Low Human Beta-Defensin 2 Gene Copy Number Predisposes to Crohn Disease of the Colon

Abstract

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 (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with ≤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 (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression. 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 (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with ≤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 (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression. Crohn disease (CD [MIM 266600]) is a severe chronic inflammatory bowel disease characterized by intestinal ulceration that affects predominantly the ileum and colon.1Loftus Jr, EV Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences.Gastroenterology. 2004; 126: 1504-1517Abstract Full Text Full Text PDF PubMed Scopus (2217) Google Scholar, 2Shanahan F Crohn’s disease.Lancet. 2002; 359: 62-69Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar The cause of the disease is unknown. 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The 50% concordance rate in MZ twins, who often exhibit the same phenotype,6Halfvarson J Bodin L Tysk C Lindberg E Jarnerot G Inflammatory bowel disease in a Swedish twin cohort: a long-term follow-up of concordance and clinical characteristics.Gastroenterology. 2003; 124: 1767-1773Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar suggests a rather balanced impact of genetic and such environmental factors as smoking7Tobin MV Logan RF Langman MJ McConnell RB Gilmore IT Cigarette smoking and inflammatory bowel disease.Gastroenterology. 1987; 93: 316-321PubMed Google Scholar, 8Bridger S Lee JC Bjarnason I Jones JE Macpherson AJ In siblings with similar genetic susceptibility for inflammatory bowel disease, smokers tend to develop Crohn’s disease and non-smokers develop ulcerative colitis.Gut. 2002; 51: 21-25Crossref PubMed Scopus (113) Google Scholar or childhood hygiene.9Gent AE Hellier MD Grace RH Swarbrick ET Coggon D Inflammatory bowel disease and domestic hygiene in infancy.Lancet. 1994; 343: 766-767Abstract PubMed Scopus (303) Google Scholar During a genomewide search, several susceptibility loci and genes—including NOD2 (CARD15) (MIM 605956),10Hugot JP Chamaillard M Zouali H Lesage S Cezard JP Belaiche J Almer S Tysk C O’Morain CA Gassull M Binder V Finkel Y Cortot A Modigliani R Laurent-Puig P Gower-Rousseau C Macry J Colombel JF Sahbatou M Thomas G Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.Nature. 2001; 411: 599-603Crossref PubMed Scopus (4480) Google Scholar, 11Ogura 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 A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.Nature. 2001; 411: 603-606Crossref PubMed Scopus (3986) Google ScholarDLG5 (MIM 604090),12Stoll 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 Genetic variation in DLG5 is associated with inflammatory bowel disease.Nat Genet. 2004; 36: 476-480Crossref PubMed Scopus (401) Google Scholar and OCTN (MIM 604190 and 603377)13Peltekova 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 Functional variants of OCTN cation transporter genes are associated with Crohn disease.Nat Genet. 2004; 36: 471-475Crossref PubMed Scopus (659) Google Scholar—have been found to be associated with CD. The best replicated is NOD2, which is involved in intracellular sensing of bacterial muramyl dipeptide, prominently expressed in macrophages and in particular small intestinal Paneth cells.14Lala S Ogura Y Osborne C Hor SY Bromfield A Davies S Ogunbiyi O Nunez G Keshav S Crohn’s disease and the NOD2 gene: a role for Paneth cells.Gastroenterology. 2003; 125: 47-57Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 15Ogura Y Lala S Xin W Smith E Dowds TA Chen FF Zimmermann E Tretiakova M Cho JH Hart J Greenson JK Keshav S Nunez G Expression of NOD2 in Paneth cells: a possible link to Crohn’s ileitis.Gut. 2003; 52: 1591-1597Crossref PubMed Scopus (357) Google Scholar Despite these significant advances, the multiple susceptibility loci and other genetic factors hitherto identified16Vermeire S Rutgeerts P Current status of genetics research in inflammatory bowel disease.Genes Immun. 2005; 6: 637-645PubMed Google Scholar 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 penetrating disease). Therefore, these behavior parameters were used in the Vienna classification,17Gasche C Scholmerich J Brynskov J D’Haens G Hanauer SB Irvine EJ Jewell DP Rachmilewitz D Sachar DB Sandborn WJ Sutherland LR A simple classification of Crohn’s disease: report of the Working Party for the World Congresses of Gastroenterology, Vienna 1998.Inflamm Bowel Dis. 2000; 6: 8-15Crossref PubMed Scopus (1000) Google Scholar recently modified in Montreal.18Silverberg MS Satsangi J Ahmad T Arnott ID Bernstein CN Brant SR Caprilli R Colombel JF Gasche C Geboes K Jewell DP Karban A Loftus Jr, EV Pena AS Riddell RH Sachar DB Schreiber S Steinhart AH Targan SR Vermeire S Warren BF Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a working party of the 2005 Montreal World Congress of Gastroenterology.Can J Gastroenterol Suppl A. 2005; 19: 5-36Google Scholar Location of disease involvement proved to be stable over time in individual patients, although the biological basis of small- versus large-intestinal involvement is unclear. NOD2 mutations have been shown to be predominantly 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).19Wehkamp J Harder J Weichenthal M Schwab M Schaffeler E Schlee M Herrlinger KR Stallmach A Noack F Fritz P Schroder JM Bevins CL Fellermann K Stange EF NOD2 (CARD15) mutations in Crohn’s disease are associated with diminished mucosal α-defensin expression.Gut. 2004; 53: 1658-1664Crossref PubMed Scopus (655) Google Scholar 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.5Fellermann K Wehkamp J Herrlinger KR Stange EF Crohn’s disease: a defensin deficiency syndrome?.Eur J Gastroenterol Hepatol. 2003; 15: 627-634Crossref PubMed Scopus (147) Google Scholar, 20Wehkamp J Schmid M Fellermann K Stange EF Defensin deficiency, intestinal microbes, and the clinical phenotypes of Crohn’s disease.J Leukoc Biol. 2005; 77: 460-465Crossref PubMed Scopus (135) Google Scholar 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-4.21Wehkamp J Harder J Weichenthal M Müller O Herrlinger KR Fellermann K Schröder JM Stange EF Inducible and constitutive b-defensins are differentially expressed in Crohn’s disease and ulcerative colitis.Inflamm Bowel Dis. 2003; 9: 215-223Crossref PubMed Scopus (248) Google Scholar, 22Fahlgren A Hammarstrom S Danielsson A Hammarstrom ML Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.Clin Exp Immunol. 2003; 131: 90-101Crossref PubMed Scopus (171) Google Scholar, 23Fahlgren A Hammarstrom S Danielsson A Hammarstrom ML β-Defensin-3 and -4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis.Clin Exp Immunol. 2004; 137: 379-385Crossref PubMed Scopus (114) Google Scholar This relative deficiency of several beta-defensins is unlikely to be explained by multiple coincident mutations or other genetic alterations of all these genes. Detailed studies of the defensin locus on chromosome 8 have uncovered an extensive DNA copy number polymorphism (CNP) of a gene cluster, including, among others, the human beta-defensin genes HBD-2 (DEFB4), HBD-3 (DEFB103), and HBD-4 (DEFB104).24Hollox EJ Armour JAL Barber JCK Extensive normal copy number variation of a β-defensin antimicrobial-gene cluster.Am J Hum Genet. 2003; 73: 591-600Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar It has been shown that the number of gene copies is positively correlated with the expression of HBD-2 in leukocytes.24Hollox EJ Armour JAL Barber JCK Extensive normal copy number variation of a β-defensin antimicrobial-gene cluster.Am J Hum Genet. 2003; 73: 591-600Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar Since all these defensins were found to be coordinately underexpressed in colonic CD,21Wehkamp J Harder J Weichenthal M Müller O Herrlinger KR Fellermann K Schröder JM Stange EF Inducible and constitutive b-defensins are differentially expressed in Crohn’s disease and ulcerative colitis.Inflamm Bowel Dis. 2003; 9: 215-223Crossref PubMed Scopus (248) Google Scholar we hypothesized that this particular phenotype may be associated with a low beta-defensin gene cluster copy number. We therefore measured DNA copy number in the beta-defensin cluster in two independent cohorts with inflammatory bowel diseases and related it to mucosal HBD-2 gene expression. 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 inpatient centers. Patients gave their informed consent, and local ethics committees approved the study protocols. A small cohort—of patients with colonic CD (n=10) and healthy control individuals (n=10)—was recruited in Stuttgart. Their blood-derived 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 confirmatory 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). Diagnostic and (Vienna) classification criteria were the same at both European centers.17Gasche C Scholmerich J Brynskov J D’Haens G Hanauer SB Irvine EJ Jewell DP Rachmilewitz D Sachar DB Sandborn WJ Sutherland LR A simple classification of Crohn’s disease: report of the Working Party for the World Congresses of Gastroenterology, Vienna 1998.Inflamm Bowel Dis. 2000; 6: 8-15Crossref PubMed Scopus (1000) Google Scholar 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 (n=103), individuals unaffected by inflammatory bowel disease who underwent surveillance colonoscopy (n=46), and 20 control individuals unaffected by intestinal disease from Cleveland. All colonoscopies that included biopsies were performed on patients from the Stuttgart cohort.Table 1Number of Patients in the Cohorts Examined for HBD-2 Gene Copy NumberNo. of SubjectsCohortClevelandStuttgartViennaControl20149CD:8554111 Ileal (L1)602238 Colonic (L2)251036 Ileal and colonic (L3)2237UC3837 Open table in a new tab DNA preparation, labeling, hybridization, and analysis procedures were performed as published elsewhere.25Mendrzyk F Korshunov A Toedt G Schwarz F Korn B Joos S Hochhaus A Schoch C Lichter P Radlwimmer B Isochromosome breakpoints on 17p in medulloblastoma are flanked by different classes of DNA sequence repeats.Genes Chromosomes Cancer. 2006; 45: 401-410Crossref PubMed Scopus (38) Google Scholar, 26Stange DE Radlwimmer B Schubert F Traub F Pich A Toedt G Mendrzyk F Lehmann U Eils R Kreipe H Lichter P High-resolution genomic profiling reveals association of chromosomal aberrations on 1q and 16p with histologic and genetic subgroups of invasive breast cancer.Clin Cancer Res. 2006; 12: 345-352Crossref PubMed Scopus (72) Google Scholar 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.27Mendrzyk F Radlwimmer B Joos S Kokocinski F Benner A Stange DE Neben K Fiegler H Carter NP Reifenberger G Korshunov A Lichter P Genomic and protein expression profiling identifies CDK6 as novel independent prognostic marker in medulloblastoma.J Clin Oncol. 2005; 23: 8853-8862Crossref PubMed Scopus (186) Google Scholar For the beta-defensin locus at 8p23.1, all additional genomic fragments that were available at the time were added to the microarray 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 >3; mean/median intensity <1.3; SD of genomic fragment log ratios <0.25) and were normalized blockwise. Chromosomal breakpoints delimiting regions of different copy number status were detected by GLAD (gain and loss analysis of DNA).28Hupe P Stransky N Thiery JP Radvanyi F Barillot E Analysis of array CGH data: from signal ratio to gain and loss of DNA regions.Bioinformatics. 2004; 20: 3413-3422Crossref PubMed Scopus (427) Google Scholar A TaqMan real-time PCR assay, specifically for amplification of genomic HBD-2, was established by using a specific set of amplification 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.29Schaeffeler E Schwab M Eichelbaum M Zanger UM CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-time PCR.Hum Mutat. 2003; 22: 476-485Crossref PubMed Scopus (137) Google Scholar 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 system. Amplification reactions (25 μl each) were performed in triplicate 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 albumin in a one-tube biplex assay, limiting primer conditions were identified, to avoid competition of the two reactions. Quantification was performed by both the standard-curve method and the comparative CT (threshold cycle) method, as described elsewhere.29Schaeffeler E Schwab M Eichelbaum M Zanger UM CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-time PCR.Hum Mutat. 2003; 22: 476-485Crossref PubMed Scopus (137) Google Scholar The assay was validated with a selection of DNA samples, genotyped elsewhere (kindly provided by E. J. Hollox, Nottingham, United Kingdom), that contained 3, 4, 5, and 7 HBD-2 gene copies.24Hollox EJ Armour JAL Barber JCK Extensive normal copy number variation of a β-defensin antimicrobial-gene cluster.Am J Hum Genet. 2003; 73: 591-600Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar Frozen biopsies were disrupted in 1 ml of Trizol (Gibco BRL) until complete fragmentation occurred. Total RNA was extracted according to the supplier’s protocol. RNA quality was determined by electrophoresis and was quantified by photometry. Subsequently, 2 μg of RNA was reverse transcribed with oligo-dT primers and 200 U Superscript (Gibco BRL), according to the routine procedure. cDNA samples were subjected to real-time PCR as outlined elsewhere.21Wehkamp J Harder J Weichenthal M Müller O Herrlinger KR Fellermann K Schröder JM Stange EF Inducible and constitutive b-defensins are differentially expressed in Crohn’s disease and ulcerative colitis.Inflamm Bowel Dis. 2003; 9: 215-223Crossref PubMed Scopus (248) Google Scholar In brief, an aliquot corresponding to 50 ng of RNA was set up in a 20-μl reaction mixture containing 4 mM MgCl2, 0.5 μM of each primer (for HBD-2, forward 5′-ATCAGCCATGAGGGTCTTGT-3′ and reverse 5′-GAGACCACAGGTGCCAATTT-3′ [annealing temperature 60°C; product 172 bp]; for HBD-3, forward 5′-TGAAGCCTAGCAGCTATGAGGATC-3′ and reverse 5′- CCGCCTCTGACTCTGCAATAA-3′ [annealing temperature 62°C; product 128 bp]; for interleukin 8 [IL-8 (MIM 146930)], forward 5′-ATGACTTCCAAGCTGGCCGTGGC-3′ and reverse 5′-TCTCAGCCCTCTTCAAAAACTTC-3′ [annealing temperature touch down protocol 66°C–60°C; product 292 bp] [Sigma]) and 1× LightCycler-FastStart DNA Master SYBR Green I Mix (Roche Molecular Biochemicals) 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). Exons 1 and 2 of the HBD2 gene and ∼50 bp of adjacent noncoding 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. 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 distribution among the clinical cohorts were assessed by the Pearson χ2 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. Using an array CGH with ∼8,000 genomic fragments covering 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 variations. 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 sequence 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-defensin 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 number gains (fig. 1D). In control individuals, no such bias was observed (fig. 1E). The neighboring alpha-defensin gene 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 search30Schutte BC Mitros JP Bartlett JA Walters JD Jia HP Welsh MJ Casavant TL McCray Jr, PB Discovery of five conserved β-defensin gene clusters using a computational search strategy.Proc Natl Acad Sci USA. 2002; 99: 2129-2133Crossref PubMed Scopus (425) Google Scholar—did not show any copy number variation in patients or control individuals (data not shown). 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. Furthermore, 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.25Mendrzyk F Korshunov A Toedt G Schwarz F Korn B Joos S Hochhaus A Schoch C Lichter P Radlwimmer B Isochromosome breakpoints on 17p in medulloblastoma are flanked by different classes of DNA sequence repeats.Genes Chromosomes Cancer. 2006; 45: 401-410Crossref PubMed Scopus (38) Google Scholar Therefore, we decided, as an alternative, to use quantitative 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 population and to two patient cohorts, one from the United States and one from Europe (table 1). In the control population (n=169) the copy numbers had a range of 2–10 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 (>4) number of copies were about equal. Details of the copy number frequencies of all cohorts and subgroups are given in table 2.Table 2HBD-2 Gene Copy Number FrequenciesPercentage of Cohort with HBD-2 Gene Copy Number ofCohort2345678910Control: Cleveland5.015.045.025.05.05.0 Europe2.723.538.323.510.1.7.7.7Cleveland: Ileal1.736.735.020.06.7 Colonic12.060.016.08.04.0European: L15.023.343.321.71.75.0 L22.250.032.610.92.22.2 L38.532.235.615.35.13.4 UC5.333.333.314.79.32.71.3Note.—For the number of patients in these subgroups, see table 1. Open table in a new tab Note.— For the number of patients in these subgroups, see table 1. The U.S. patient cohort from the Cleveland Clinic consisted of 85 surgical patients with CD who had indications for ileal versus colonic resection. In patients with ileal resections (fig. 2B), the median number of copies was identical to that of the control group (4 copies); also, the frequency distribution of the three subgroups (with <4, with 4, and with >4 gene copy numbers) was not significantly 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 (P=.008 [Mann-Whitney]) between the ileal and colonic subgroups with CD. This was maintained when tested by Kruskal-Wallis analysis of variance (P<.01 for both post 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 (P=.018 [Pearson χ2]). An independent second cohort of European patients with CD (n=165) from Stuttgart and Vienna was classified according 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 (P>.05), with a majority in the group with 4 gene copies (fig. 3). The median was 4 copies in L1 and controls compared with 3 in L2 (P=.032 and P=.001, respectively [Mann-Whitney]). Analysis of variance and post hoc test identified a significant difference between L2 and controls only (P<.05). Colonic CD only (L2) was characterized by a shift to lower copy numbers, with the majority (52%) carrying <4 copies of HBD-2 (P=.002 vs. controls; P=.037 vs. L1 [Pearson χ2]). Individuals with ≤3 copies have 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 distribution pattern between controls and L2, and, although the median was the normal 4 copies, the shift in distribution was significant (P=.034 [Mann-Whitney]