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 < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 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.

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.

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 >4 copies per genome. The difference in distribution between ileal and colonic resection was significant (P=.018 [χ² test]).

Figure 3.

Distribution of HBD-2 gene copy numbers (<4, 4, or >4 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 (P=.037 and P=.002, respectively [χ² test]).

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.