Genomic copy number determines functional expression of {beta}-defensin 2 in airway epithelial cells and associates with chronic obstructive pulmonary disease.

RATIONALE: Copy number variations of the cluster of beta-defensin genes have been associated with psoriasis and inflammatory bowel disease. Controversy still exists on whether the beta-defensins genes determine susceptibility for chronic obstructive pulmonary disease (COPD). OBJECTIVES: We investigated whether genomic copy number variations of the beta-defensin gene cluster have a functional role in airway epithelial cells and associate with the presence of COPD. METHODS: Baseline and inflammatory induced transcript expression of DEFB4 was studied in nasal epithelial cell cultures and its effect on Pseudomonas aeruginosa inhibition was assessed. Subsequently, relevant functional cut-offs for copy numbers were used to explore associations with COPD in two independent case-control studies. MEASUREMENTS AND MAIN RESULTS: Copy number variation in the beta-defensin encoding genes correlated with baseline mRNA DEFB4 expression levels (R(2) = 0.96; P = 0.02), with a plateau effect from five copies or more. Only when higher copy numbers of beta-defensin genes were present, transcription was significantly up-regulated by tumor necrosis factor-alpha (P < 0.0001), which resulted in better antimicrobial activity in vitro. When comparing healthy smokers with COPD patients, a copy number greater than or equal to 5 was associated with increased risk for COPD with an adjusted odds ratio of 1.8 (confidence interval, 1.1-2.8; P = 0.02), which was confirmed by a second independent case-control study. CONCLUSIONS: Genomic copy number variation of beta-defensin encoding genes has a functional role in airway epithelial cells, which may contribute to the pathogenesis of COPD.

Accurate determination of copy number variations (CNVs): application to the alpha- and beta-defensin CNVs.

The human genome is rich in genomic regions that are repeated several times. These regions are known as CNVs (copy number variations) and can be polymorphic. Moreover, the number of copies may play a role in the predisposition to particular diseases. It is therefore important to accurately determine the copy number of those CNVs in individuals. We have developed a strategy, using concatemeric constructs containing different numbers of repeats as internal standards, to accurately determine the number of repeats in the alpha-defensin and beta-defensin region on chromosome 8p23 by real-time PCR. The test was validated by FISH in DNA of 13 individuals. Comparison with previously published methods shows that this approach provides more accurate results for the determination of the exact number of repeats when they exceed 3 copies. This strategy can be easily transferred to any CNV. With this method we structurally analyzed the alpha- and beta-defensin region in 334 Belgian individuals.

Cystic fibrosis transmembrane conductance regulator gene polymorphisms in patients with primary sclerosing cholangitis.

BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is a progressive cholestatic disease commonly associated with inflammatory bowel disease (IBD) and characterized by fibrosing inflammatory destruction of bile ducts. The histological features in the liver of PSC patients are similar to those observed in cystic fibrosis (CF). Our aim was to study whether variants in the CFTR gene are associated with the occurrence and/or evolution of PSC. METHODS: PSC patients (n=140) were genotyped for F508del, the TGmTn variants, and four additional polymorphic loci (1001+11 C>T, M470V, T854T and Q1463Q), and compared to 136 matched healthy controls. RESULTS: The 1540G-allele, encoding V470, was less frequent in PSC (52%) than in controls (64%, p=0.003), and was associated with protection against PSC in individuals without IBD (OR 0.25, 95% CI 0.12-0.52, p=0.0002). Also TG11-T7 was less frequent in PSC (53%) than in controls (61%, p=0.04), this haplotype was associated with reduced risk for PSC (OR 0.34, 95% CI 0.17-0.70, p=0.003) in individuals without IBD. CONCLUSIONS: In this cohort of PSC patients, several CFTR-variants affecting the functional properties of the CFTR protein seem to offer protection against the development of PSC, confirming our hypothesis that CFTR might be implicated in the pathogenesis of PSC.

Differential induction of human beta-defensin expression by periodontal commensals and pathogens in periodontal pocket epithelial cells.

BACKGROUND: To investigate the possible role of beta-defensins in gingival health and periodontal disease, we examined the effect of several stimuli on the expression of interleukin-8 (IL-8), human beta-defensin-1, -2, -3, and -4 (hBD) in primary human diseased gingival epithelial (HGE) cell cultures from periodontitis patients by quantitative TaqMan reverse transcription polymerase chain reaction (RT-PCR). METHODS: Several strains of the periodontopathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were added to the cells, as well as the oral commensal bacteria Fusobacterium nucleatum and Escherichia coli. The induction by the proinflammatory stimuli phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-alpha (TNF-alpha) was also tested. RESULTS: In addition to the published observations (PMA induces hBD-2 and -4; TNF-alpha induces hBD-2 and -3), it was found that PMA can upregulate hBD-1 and hBD-3, whereas TNF-alpha can induce hBD-4. The commensal bacteria were significant inducers of hBD-2, hBD-3, and IL-8. The pathogen P. gingivalis induced hBD-1 and hBD-3 at different time points than the commensals, but no induction of IL-8 and hBD-2 could be observed. These data fit with the chemokine paralysis theory. A correlation was found between the pathogenicity of different serotypes of A. actinomycetemcomitans and the induction profiles of defensins and IL-8. CONCLUSION: The results suggest that a correlation can be found in diseased oral epithelium between the defensin profiles that are induced and the pathogenicity of the oral bacterial strains.

Distribution of human beta-defensin polymorphisms in various control and cystic fibrosis populations.

Human beta defensins contribute to the first line of defense against infection of the lung. Polymorphisms in these genes are therefore potential modifiers of the severity of lung disease in cystic fibrosis. Polymorphisms were sought in the human beta-defensin genes DEFB1, DEFB4, DEFB103A, and DEFB104 in healthy individuals and cystic fibrosis (CF) patients living in various European countries. DEFB1, DEFB4, and DEFB104 were very polymorphic, but DEFB103A was not. Within Europe, differences between control populations were found for some of the frequent polymorphisms in DEFB1, with significant differences between South-Italian and Czech populations. Moreover, frequent polymorphisms located in DEFB4 and DEFB104 were not in Hardy Weinberg equilibrium in all populations studied, while those in DEFB1 were in Hardy Weinberg equilibrium. Sequencing of a monochromosomal chromosome 8 mouse-human hybrid cell line revealed signals for multiple alleles for some loci in DEFB4 and DEFB104, but not for DEFB1. This indicated that more than one DEFB4 and DEFB104 gene was present on this chromosome 8, in agreement with recent findings that DEFB4 and DEFB104 are part of a repeat region. Individual DEFB4 and DEFB104 PCR amplification products of various samples were cloned and sequenced. The results showed that one DNA sample could contain more than two haplotypes, indicating that the various repeats on one chromosome were not identical. Given the higher complexity found in the genomic organization of the DEFB4 and DEFB104 genes, association studies with CF lung disease severity were performed only for frequent polymorphisms located in DEFB1. No association with the age of first infection by Pseudomonas aeruginosa or with the FEV1 percentage at the age of 11-13 years could be found.