Screening of copy number polymorphisms in human beta-defensin genes using modified real-time quantitative PCR

Defensins are cationic antimicrobial peptides, which play an important role in host immune defense to some infectious diseases as well as immune disease and skin disease. Recent studies identified that the genes coding for human beta-defensin 2 (DEFB4), human beta-defensin 3 (DEFB103) and human beta-defensin 4 (DEFB104) showed variation in copy numbers. This variation may have an impact on gene expression levels. Here, we have demonstrated a real-time PCR-based method to measure beta-defensin gene copy number. Using this relative real-time quantitative PCR, we developed a new rapid and reliable approach, which involves amplification of the target locus (DEFB4 or DEFB103 or DEFB104) and the single-copy reference locus (human serum albumin, ALB) in a single PCR reaction. A calibrator was prepared by recombining one copy of the target gene and one copy of the reference gene into a plasmid. After correcting the PCR amplification efficiency, which differed between the defensin gene and ALB gene, and normalization by the calibrator, the ratio of the copy number of the target gene to that of the reference gene in an unknown sample was determined. This normalized ratio directly related to the gene copy number. The assay was validated using previously genotyped samples, which demonstrated high accuracy and reliability of the method. Furthermore, this method was used to screen the copy number variations of these three beta-defensin genes in healthy blood donors. This method proved to be a reliable and fast tool to genotype gene copy number variations in projects associating genomic variations with gene expression or with population phenotypes in epidemiologic studies.     

Association of beta-defensin-1 gene polymorphisms with Pseudomonas aeruginosa airway colonization in cystic fibrosis

Lung disease and Pseudomonas aeruginosa (P. aeruginosa) airway colonization represent a major cause of morbidity and mortality in cystic fibrosis (CF). Human beta-defensin (hBD)-1 is believed to play an important role in mucosal innate immunity in the lung. This study aimed to investigate whether three single-nucleotide polymorphisms (SNPs) in the 5'-untranslated region of DEFB1, G-52A, C-44G and G-20A were associated with P. aeruginosa airway colonization in CF. A total of 224 CF patients and 196 control subjects were studied. DEFB1 SNPs were characterized by restriction fragment length polymorphisms. Patients' sputum samples were collected and analyzed by standard methods. Single SNP analysis suggested that CF patients carrying the -52AA and the -20GG genotypes had a higher rate of P. aeruginosa airway colonization than patients homozygous and heterozygous for the -52G and -20A alleles (P=0.01 and P=0.007, respectively). A significant association between the ACG haplotype and chronic P. aeruginosa infection was also identified (odds ratio (95% confidence interval): 3.00 (1.42-6.36), P=0.004). These results indicate that variant alleles in DEFB1 might contribute to the colonization of P. aeruginosa in CF.     

Analysis of DEFB1 regulatory SNPs in cystic fibrosis patients from North-Eastern Italy

Cystic fibrosis (CF) transmembrane regulator protein (CFTR) gene is undoubtedly the main genetic factor involved in the modulation of CF phenotype. However, other factors such as human defensins and the genes encoding for these antimicrobial peptides have been hypothesized as possible modifiers influencing airways infection in CF patients, but their role in the pathogenesis of lung disease is still debated. Since DEFB1 gene encoding for human beta-defensin 1 displays features such as antimicrobial or chemotactic activity playing a role in inflammation, it has been considered as a possible candidate CF modifier gene. We analysed three single nucleotide polymorphisms (SNPs) in the 5′-untranslated region of the DEFB1 gene (namely g-52G>A, g-44C>G and g-20G>A) in a group of 62 CF patients from North Eastern Italy, and in 130 healthy controls, with the aim of verifying the possible association of these functional SNPs with the pulmonary phenotype of CF patients. DEFB1 SNPs have been genotyped by using Taqman allele-specific fluorescent probes and a real-time PCR platform. No significant differences were found for allele, genotype and haplotype frequencies of DEFB1 g-52G>A, g-44C>G and g-20G>A SNPs in CF patients stratified for Pseudomonas aeruginosa infection, as well as in patients with a severe and mild clinical phenotype or in patients stratified for CFTR genotypes. DEFB1 allele, genotype and haplotype frequencies of CF patients globally considered were similar to those of healthy controls. Our findings are discordant with respect to another recent study performed on CF patients coming from Southern Italy, probably due to different ethnicity of the patients.     

A worldwide analysis of beta-defensin copy number variation suggests recent selection of a high-expressing DEFB103 gene copy in East Asia

Beta-defensins are a family of multifunctional genes with roles in defense against pathogens, reproduction, and pigmentation. In humans, six beta-defensin genes are clustered in a repeated region which is copy-number variable (CNV) as a block, with a diploid copy number between 1 and 12. The role in host defense makes the evolutionary history of this CNV particularly interesting, because morbidity due to infectious disease is likely to have been an important selective force in human evolution, and to have varied between geographical locations. Here, we show CNV of the beta-defensin region in chimpanzees, and identify a beta-defensin block in the human lineage that contains rapidly evolving noncoding regulatory sequences. We also show that variation at one of these rapidly evolving sequences affects expression levels and cytokine responsiveness of DEFB103, a key inhibitor of influenza virus fusion at the cell surface. A worldwide analysis of beta-defensin CNV in 67 populations shows an unusually high frequency of high-DEFB103-expressing copies in East Asia, the geographical origin of historical and modern influenza epidemics, possibly as a result of selection for increased resistance to influenza in this region.     

High-resolution mapping of the 8p23.1 beta-defensin cluster reveals strictly concordant copy number variation of all genes

One unexpected feature of the human genome is the high structural variability across individuals. Frequently, large regions of the genome show structural polymorphisms and many vary in their abundance. However, accurate methods for the characterization and typing of such copy number variations (CNV) are needed. The defensin cluster at the human region 8p23.1 is one of the best studied CNV regions due to its potential clinical relevance for innate immunity, inflammation, and cancer. The region can be divided into two subclusters, which harbor predominantly either alpha- or beta-defensin genes. Previous studies assessing individual copy numbers gave different results regarding whether the complete beta-defensin cluster varies or only particular genes therein. We applied multiplex ligation-dependent probe amplification (MLPA) to measure defensin locus copy numbers in 42 samples. The data show strict copy number concordance of all 10 loci typed within the beta-defensin cluster in each individual, while seven loci within the alpha-defensin cluster are consistently found as single copies per chromosome. The exception is DEFA3, which is located within the alpha-defensin cluster and was found to also differ in copy number interindividually. Absolute copy numbers ranged from two to nine for the beta-defensin cluster and zero to four for DEFA3. The CNV-typed individuals, including HapMap samples, are publicly available and may serve as a universal reference for absolute copy number determination. On this basis, MLPA represents a reliable technique for medium- to high-throughput typing of 8p23.1 defensin CNV in association studies for diverse clinical phenotypes.     

ORFeome-based search of airway epithelial cell-specific novel human [beta]-defensin genes

beta-Defensin is one of the major host defense shields produced by various tissues and organs against microbial infection. To date, four human beta-defensins (DEFBs) gene products that share a consensus six-cysteine motif have been discovered. The hidden Markov model (HMM) profile was constructed from the common features of those known beta-defensin peptides to search for additional novel DEFB genes. A genome-wide search of the profile against ORFeome-based peptide databases (e.g., Ensembl project) led to the identification of six new DEFB members that also shared the conserved six-cysteine motif. Phylogenetic analysis supported a close relationship of these six new members with existing DEFB genes. Polymerase Chain Reaction studies of human tissue cDNA panels confirmed the expression of all six novel DEFB genes in various tissues. Two of them, DEFB106 and DEFB109, were expressed in the lung. A pilot study with cRNA probes for in situ hybridization and a synthetic propeptide for the functional characterization demonstrated the tissue-/cell-specific expression and the strong antimicrobial activity of DEFB106. These results support the utility of ORFeome-based HMM search in gene discovery for members of a specific gene family. The novel DEFB genes identified in this study may significantly contribute to overall antimicrobial host defenses.     

Association of beta-defensin 1 single nucleotide polymorphisms with atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is a chronic multifactorial allergic disease with unclear etiology. The antimicrobial human beta-defensin 1 is chemotactic for dendritic cells, which are important regulators of allergic immune responses. In an attempt to identify useful markers that could predict susceptibility to AD, we investigated single nucleotide polymorphisms (SNPs) of the beta-defensin 1 gene (DEFB1) with potential functional consequences. METHODS: Four SNPs of the DEFB1 gene were genotyped either by real-time polymerase chain reaction or polymerase chain reaction-restriction fragment length polymorphisms in 59 patients with AD and 151 controls from the Mexican population. Correlation analyses were carried out between genetic, environmental and clinical variables in AD patients. RESULTS: The genotypes associated with susceptibility to AD and no other allergy were 692 GG (OR = 3.21, 95% CI 1.37-7.34) and 1654 AA (OR = 17.37, 95% CI 1.62-860.83). The allele 668 C is a risk factor for AD (OR = 2.23, 95% CI 1.22-4.01) and the allele A in site 1836 correlates with earlier age at onset (Spearman's rho = 0.232; p = 0.03). The prolonged duration of breastfeeding correlates with earlier age at onset as well as with the severity of AD. CONCLUSIONS: The DEFB1 gene is probably involved in the incidence and development of AD, but additional functional studies will be necessary to understand the biological role of these SNPs.     

Variation in human β‐defensin genes: new insights from a multi‐population study

Human β‐defensin 2 (hBD‐2) and hBD‐3, encoded by DEFB4 and DEFB103A, respectively, have shown anti‐HIV activity, and both genes exhibit copy number variation (CNV). Although the role of hBD‐1, encoded by DEFB1, in HIV‐1 infection is less clear, single nucleotide polymorphisms (SNPs) in DEFB1 may influence viral loads and disease progression. We examined the distribution of DEFB1 SNPs and DEFB4/103A CNV, and the relationship between DEFB1 SNPs and DEFB4/103A CNV using samples from two HIV/AIDS cohorts from the United States (n = 150) and five diverse populations from the Coriell Cell Repositories (n = 46). We determined the frequencies of 10 SNPs in DEFB1 using a post‐PCR, oligonucleotide ligation detection reaction–fluorescent microsphere assay, and CNV in DEFB4/103A by real‐time quantitative PCR. There were noticeable differences in the frequencies of DEFB1 SNP alleles and haplotypes among various racial/ethnic groups. The DEFB4/103A copy numbers varied from 2 to 8 (median, 4), and there was a significant difference between the copy numbers of self‐identified whites and blacks in the US cohorts (Mann–Whitney U‐test P = 0.04). A significant difference was observed in the distribution of DEFB4/103A CNV among DEFB1 ‐52G/A and ‐390T/A genotypes (Kruskal–Wallis P = 0.017 and 0.026, respectively), while not in the distribution of DEFB4/103A CNV among ‐52G/A_‐44C/G_‐20G/A diplotypes. These observations provide additional insights for further investigating the complex interplay between β‐defensin genetic polymorphisms and susceptibility to, or the progression or severity of, HIV infection/disease.     

Fundamental role for HIF-1α in constitutive expression of human β defensin-1

Antimicrobial peptides are secreted by the intestinal epithelium to defend from microbial threats. The role of human β defensin-1 (hBD-1) is notable because its gene (beta-defensin 1 (DEFB1)) is constitutively expressed and its antimicrobial activity is potentiated in the low-oxygen environment that characterizes the intestinal mucosa. Hypoxia-inducible factor (HIF) is stabilized even in healthy intestinal mucosa, and we identified that epithelial HIF-1α maintains expression of murine defensins. Extension to a human model revealed that basal HIF-1α is critical for the constitutive expression of hBD-1. Chromatin immunoprecipitation identified HIF-1α binding to a hypoxia response element in the DEFB1 promoter whose importance was confirmed by site-directed mutagenesis. We used 94 human intestinal samples to identify a strong expression correlation between DEFB1 and the canonical HIF-1α target GLUT1. These findings indicate that basal HIF-1α is critical for constitutive expression of enteric DEFB1 and support targeting epithelial HIF for restoration and maintenance of intestinal integrity.     

Genomic variations within DEFB1 are associated with the susceptibility to and the fatal outcome of severe sepsis in Chinese Han population

Sepsis is a systemic inflammatory response syndrome to infection. Human beta-defensin 1 (DEFB1) is a multifunctional mediator in infection and inflammation, which has been largely explored in ex vivo studies. The present case-control study was designed to investigate whether DEFB1 genomic variations are associated with the susceptibility to and the outcome of severe sepsis in 211 patients with severe sepsis and 157 ethnic-matched healthy controls. After correcting for multiple testing, the -44G/C was the only polymorphism found to show significant associations with both the susceptibility to and the fatal outcome of severe sepsis (P=0.0049, odd ratio (OR) 1.971 and P=0.002, OR 2.406, respectively). Haplotype -20A/-44C/-52G showed a protective role against severe sepsis (P=0.0066, OR 0.6751), whereas haplotype -20G/-44G/-52G served as a risk factor for the fatal outcome of severe sepsis (P=0.0052, OR 2.427). These findings provide further evidence that beta-defensin 1 may play a role in the pathogenesis of severe sepsis.     

Correlating Multiallelic Copy Number Polymorphisms with Disease Susceptibility

The human genome contains a significant amount of sequence variation, from single nucleotide polymorphisms to large stretches of DNA that may be present in a range of different copies between individuals. Several such regions are variable in >1% of the population (referred to as copy number polymorphisms or CNPs), and many studies have looked for associations between the copy number of genes within multiallelic CNPs and disease susceptibility. Associations have indeed been described for several genes, including the β‐defensins (DEFB4, DEFB103, DEFB104), chemokine ligand 3 like 1 (CCL3L1), Fc gamma receptor 3B (FCGR3B), and complement component C4 (C4). However, follow‐up replication in independent cohorts has failed to reproduce a number of these associations. It is clear that replicated associations such as those between C4 and systemic lupus erythematosus, and β‐defensin and psoriasis, have used robust genotyping methodologies. Technical issues associated with genotyping sequences of high identity may therefore account for failure to replicate other associations. Here, we compare and contrast the most popular approaches that have been used to genotype CNPs, describe how they have been applied in different situations, and discuss potential reasons for the difficulty in reproducibly linking multiallelic CNPs to complex diseases.     

Asthma and atopy are associated with DEFB1 polymorphisms in Chinese children

Human beta-defensin (HBD)-1 is constitutively expressed in the airway, and hBD-1 plays crucial roles in innate immunity against respiratory pathogens. Asthma was associated with DEFB1 polymorphisms in Caucasians. This study investigates whether three single nucleotide polymorphisms (SNPs) in 5'-untranslated region of DEFB1 are associated with asthma phenotypes in Chinese children. Subjects aged 5-18 years were recruited from general pediatric clinics. Plasma IgE concentrations were measured by immunoassays. DEFB1 SNPs were characterized by restriction fragment length polymorphism. In all, 305 asthmatics and 156 controls were recruited. For asthma diagnosis, atopy and plasma total IgE, higher percentages of subjects with these outcomes had the minor alleles -20A and -52G (P = 0.041-0.0002). For log-transformed total IgE, the covariate was positive and significant for G-20A under recessive model (P = 0.001) and for G-52A under both recessive and codominant models (P = 0.008 and 0.035). The recessive model covariate was also positive and significant (P = 0.020) for C-44G on peripheral blood eosinophil count. The GCA haplotype of DEFB1 was significantly associated with asthma (odds ratio (95% confidence interval): 1.64 (1.05-2.57); P = 0.029). These results suggest that DEFB1 is a candidate gene for asthma and atopy in children.     

Interaction between seroreactivity to microbial antigens and genetics in Crohn's disease: is there a role for defensins?

Antibodies against different microbial epitopes are associated with disease phenotype, may be of diagnostic importance and may reflect a loss of tolerance in Crohn's disease (CD). Recently, an association was reported between the presence of these antibodies and mutations in pattern receptor genes. Our aim was to investigate whether mutations in various genes other than NOD2/CARD15 or TLR4 associated with CD (NOD1/CARD4, DLG5 and DEFB1) may influence the presence of antibodies against bacterial proteins and carbohydrates in a Hungarian cohort of CD patients. Three hundred and seventy-six well-characterized, unrelated, consecutive CD patients (male/female: 191/185, age at onset: 29.1 +/- 12.9 years, duration: 7.9 +/- 11.7 years) were investigated. Sera were assayed for anti-Omp, anti-Saccharomyces cerevisiae antibodies (ASCAs) immunoglobulin (Ig) A and IgG, and antibodies against a mannan epitope of S. cerevisiae (gASCA), laminaribioside (ALCA), chitobioside (ACCA), and mannobioside (AMCA). NOD1/CARD4, DLG5 and DEFB1 variants were tested by polymerase chain reaction-restriction fragment length polymorphism, and DEFB1 was genotyped in a subgroup of 160 patients. Detailed clinical phenotypes were determined by reviewing the patients' medical charts. The carriage of DEFB1 20A variant alleles less frequently led to antiglycan positivity compared with patients without (29.6% vs 46.2%, OR: 0.49, 95% CI: 0.25-0.97), regardless of disease location or behavior. Similar tendency was observed for DEFB1 44G (present: 21.6% vs absent: 10.2%, P = 0.06) and ALCA. A gene or serology dosage effect was not observed. However, no association was found between the DEFB1 G52A, DLG5 R30Q, and NOD1/CARD4 E266K variants and any of the serology markers. We found that variants in human beta-defensin 1 gene are inversely associated with antiglycan antibodies, further confirming an important role for innate immunity in the pathogenesis of CD.     

Copy number variation in autoimmunity — importance hidden in complexity?

Copy number variation, namely regions of the genome that can be either deleted or duplicated in a variable way, has emerged as an important source of genetic variance in the human genome. Genes with immunological functions are particularly prone to copy number variation, in part because this is a mechanism to expand the recognition repertoire; however, immunological genes not directly involved in immune recognition are also copy number variable but, despite the link between immunological function and copy number variation, very few copy number variants (CNVs) have been found to be associated with autoimmune diseases, even in recent large genome-wide CNV-association studies. Nonetheless, CNVs in FCGR3B, DEFB4, CCL3L1, C4A/B and NCF1 have been suggested to be associated with autoimmune diseases, although there is conflicting evidence in all cases. The reasons for the lack of definitive data on CNV-autoimmunity associations, as well as the technical challenges for the field are the focus of this review.     

Novel hairpin-shaped primer assay to study the association of the -44 single-nucleotide polymorphism of the DEFB1 gene with early-onset periodontal disease

A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The -44 C-->G transversion in the DEFB1 gene (which encodes human beta-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the -44 SNP. We used an HP assay to study the distribution of the -44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the -44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.     

Genotype-phenotype correlation in cystic fibrosis: the role of modifier genes

More than 1,000 mutations have been identified in the cystic fibrosis (CF) transmembrane regulator (CFTR) disease gene. The impact of these mutations on the protein and the wide spectrum of CF phenotypes prompted a series of Genotype-Phenotype correlation studies. The CFTR genotype is invariably correlated with pancreatic status-in about 85% of cases with pancreatic insufficiency and in about 15% of cases with pancreatic sufficiency. The correlations between the CFTR genotype and pulmonary, liver, and gastrointestinal expression are debatable. The heterogeneous phenotype in CF patients bearing the same genotype or homozygotes for nonsense mutations implicated environmental and/or genetic factors in the disease. However, the discordant phenotype observed in CF siblings argued against a major role of environmental factors and suggested that genes other than CFTR modulate the CF phenotype. A locus that modulates gastrointestinal expression was identified in mice and subsequently in humans. By analyzing nine CF patients discordant for meconium ileus we were able to show that this locus had a dominant effect. Moreover, in a collaborative study we found a higher rate of polymorphisms in beta-defensin genes 1 and 2 in CF patients and in controls. In another multicenter study mutations in alpha-1 antitrypsin (A1AT) and mannose binding lectin genes were found to be independent risk factors for liver disease in CF patients. The body of evidence available suggests that the variegated CF phenotype results from complex interactions between numerous gene products.     

Single-nucleotide polymorphisms (SNPs) in human beta-defensin 1: high-throughput SNP assays and association with Candida carriage in type I diabetics and nondiabetic controls

beta-Defensins are cationic antimicrobial peptides expressed in epithelia. They exhibit antibacterial, antifungal, and antiviral properties. Defensins are a component of the innate immune response, and it has been proposed that they have a protective role in the oral cavity. Previous studies have shown that human beta-defensin 1 (hBD-1) is constitutively expressed in oral epithelial cells but that expression varies between individuals. We tested the hypothesis that genetic variations in defensin peptide expression may be associated with opportunistic infections. This may be critical in the immunocompromised patient population, in which innate immune responses may have a relatively more important role. Oral Candida carriage status and the presence of six single-nucleotide polymorphisms (SNPs) in the DEFB1 gene encoding hBD-1 were evaluated in type I diabetic patients (n = 43) and nondiabetic controls (n = 50). Genomic DNA was obtained from buccal swabs. Portions of the DEFB1 gene were amplified, and each SNP was analyzed by a TaqMan assay, standardized with control DNA of known genotype. Candida carriage status was determined from unstimulated saliva on CHROMagar plating medium. A low level of Candida carriage was defined as < or = 350 CFU/ml. A high level of Candida carriage was seen in 44% of the diabetic subjects but only in 28% of the nondiabetic controls (P < 0.05). C. albicans predominated; however, diabetic subjects, especially those with high levels of carriage, showed an increased proportion of Candida glabrata and C. tropicalis. There was a strong association between an SNP in the 5' untranslated region (C-->G at position -44) and Candida carriage in both groups. Among individuals in the diabetic population who had the SNP allele 2 (G), 58% had low CFU, while 6% had high CFU. The C-->G SNP at position -44 is associated with low levels of Candida carriage. The resultant odd ratios are statistically significant for a protective effect (odd ratios, 25 for diabetic subjects and 8.5 for nondiabetic subjects). These results indicate that genetic variations in the DEFB1 gene encoding hBD-1 may have a major role in mediating and/or contributing to susceptibility to oral infection.     

Validation of chromogenic in situ hybridization for detection of EGFR copy number amplification in nonsmall cell lung carcinoma.

Epidermal growth factor receptor (EGFR) gene copy number correlates with response to tyrosine kinase inhibitors in patients with nonsmall cell lung carcinoma. Fluorescence in situ hybridization (FISH), a standard methodology to detect EGFR copy number abnormalities in nonsmall cell lung carcinoma, is limited by instrumentation and cost. Chromogenic in situ hybridization (CISH) is an emerging alternative detection technique using light microscopy, but its utility in assessing EGFR copy number in lung cancer is not established. To address the utility of CISH, we studied paraffin-embedded nonsmall cell lung carcinoma specimens from 77 Taiwanese nonsmoking women treated by surgery alone. We recorded the number of signals per tumor cell nucleus, correlated EGFR copy number by CISH with FISH results, and used receiver operating characteristics to identify cut-off points for the CISH results. Tumors were classified as adenocarcinoma (n=28), mixed adenocarcinoma with bronchioloalveolar features (n=25), bronchioloalveolar carcinoma (n=2), squamous cell carcinoma (n=15), and adenosquamous carcinoma (n=7). By FISH, 29% of cases had no amplification, 18% had low polysomy, 35% had high polysomy, and 12% had gene amplification. EGFR copy number detected by CISH highly correlated with FISH (Spearman r=0.81, P<0.0001). We determined the optimal EGFR CISH cut-off points that discriminate between no amplification and low polysomy (2.8 signals, P=0.09); no amplification plus low polysomy and high polysomy plus gene amplification (4.5 signals, P<0.0001); and high polysomy and gene amplification (7.1 signals, P=0.0003). CISH is an alternative assay to FISH in determining EGFR copy number status that may contribute to stratification of patients with nonsmall cell lung carcinoma for clinical trials and identify a subset of patients that should be treated with tyrosine kinase inhibitors.