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1.  Relevance of α-defensins (HNP1-3) and defensin β-1 in diabetes 
AIM: To investigate the genetic background of human defensin expression in type 1 and 2 diabetes.
METHODS: Associations between DEFA1/DEFA3 gene copy number polymorphism and diabetes as well as between the promoter polymorphisms of DEFB1 and diabetes were studied. The copy number variation of the DEFA1/DEFA3 genes was determined in 257 diabetic patients (117 patients with type 1 and 140 with type 2 diabetes). The control group consisted of 221 age- and gender-matched healthy blood donors. The cumulative copy numbers of the DEFA1/DEFA3 genes were detected by using quantitative PCR analysis. To evaluate the HNP 1-3 (human neutrophil peptide 1-3 or α-defensin) levels in the circulation, plasma HNP 1-3 concentrations were measured by ELISA. The expression of DEFA1/A3 in peripheral leukocytes of the diabetic patients was measured by quantitative RT PCR analysis. Three SNPs of the human DEFB1 (human defensin β-1) gene: DEFB1 G-20A (rs11362), DEFB1 C-44G (rs1800972) and DEFB1 G-52A (rs1799946) were genotyped by Custom TaqMan® Real Time PCR assay.
RESULTS: Significant differences were observed in HNP1-3 levels between the healthy subjects and both groups of diabetic patients. The mean ± SE was 28.78 ± 4.2 ng/mL in type 1 diabetes, and 29.82 ± 5.36 ng/mL in type 2 diabetes, vs 11.94 ± 2.96 ng/mL in controls; P < 0.01 respectively. There was no significant difference between patients with type 1 and type 2 diabetes in the high plasma concentrations of HNP1-3. The highest concentrations of α-defensin were found in diabetic patients with nephropathy (49.4 ± 4.8 ng/mL), neuropathy (38.7 ± 4.8 ng/mL) or cardiovascular complications (45.6 ± 1.45 ng/L). There was no significant difference in the cumulative copy numbers of DEFA1/DEFA3 genes between controls and patients, or between patients with the two types of diabetes. Comparisons of HNP 1-3 plasma level and DEFA1/A3 copy number of the same patient did not reveal significant relationship between defensin-α levels and the gene copy numbers (r2 = 0.01). Similarly, no positive correlation was observed between the copy numbers and the mRNA expression levels of DEFA1/A3. Regarding the C-44G polymorphism of DEFB1, the GG “protective” genotype was much less frequent (1%-2%) among both groups of patients than among controls (9%).
CONCLUSION: Elevated HNP1-3 levels in diabetes are independent of DEFA1/DEFA3 copy numbers, but GG genotype of C-44G SNP in DEFB1 gene may result in decreased defensin β-1 production.
doi:10.3748/wjg.v20.i27.9128
PMCID: PMC4112898  PMID: 25083086
α-defensins; HNP1-3; β-defensin 1; Diabetes; Copy number polymorphism; Single nucleotide polymorphism
2.  Copy Number Variation of the Beta Defensin Gene Cluster on Chromosome 8p Influences the Bacterial Microbiota within the Nasopharynx of Otitis-Prone Children 
PLoS ONE  2014;9(5):e98269.
As there is increasing evidence that aberrant defensin expression is related to susceptibility for infectious disease and inflammatory disorders, we sought to determine if copy number of the beta-defensin gene cluster located on chromosome 8p23.1 (DEFB107, 106, 105, 104, 103, DEFB4 and SPAG11), that shows copy number variation as a block, was associated with susceptibility to otitis media (OM). The gene DEFB103 within this complex encodes human beta defensin-3 (hBD-3), an antimicrobial peptide (AP) expressed by epithelial cells that line the mammalian airway, important for defense of mucosal surfaces and previously shown to have bactericidal activity in vitro against multiple human pathogens, including the three that predominate in OM. To this end, we conducted a retrospective case-control study of 113 OM prone children and 267 controls aged five to sixty months. We identified the copy number of the above defined beta-defensin gene cluster (DEFB-CN) in each study subject by paralogue ratio assays. The mean DEFB-CN was indistinguishable between subjects classified as OM prone based on a recent history of multiple episodes of OM and control subjects who had no history of OM (4.4±0.96 versus 4.4±1.08, respectively: Odds Ratio [OR]: 1.16 (95% CI: 0.61, 2.20). Despite a lack of direct association, we observed a statistically significant correlation between DEFB-CN and nasopharyngeal bacterial colonization patterns. Collectively, our findings suggested that susceptibility to OM might be mediated by genetic variation among individuals, wherein a DEFB-CN less than 4 exerts a marked influence on the microbiota of the nasopharynx, specifically with regard to colonization by the three predominant bacterial pathogens of OM.
doi:10.1371/journal.pone.0098269
PMCID: PMC4035277  PMID: 24867293
3.  Variation in human β-defensin genes: new insights from a multi-population study 
Summary
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 by 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.
doi:10.1111/iji.12021
PMCID: PMC3664661  PMID: 23194186
4.  8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations 
BMC Genomics  2014;15:64.
Background
The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination.
Results
We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes.
Conclusion
MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.
doi:10.1186/1471-2164-15-64
PMCID: PMC3937106  PMID: 24460793
Beta-defensin; Copy number variation; Quantitative real-time PCR; Paralog ratio tests; Multiplex ligation-dependent probe amplification; Clustering; Pseudogene; Low frequency sequence variations
5.  Copy Number Variation of the Beta-Defensin Genes in Europeans: No Supporting Evidence for Association with Lung Function, Chronic Obstructive Pulmonary Disease or Asthma 
PLoS ONE  2014;9(1):e84192.
Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02–1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72–1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.
doi:10.1371/journal.pone.0084192
PMCID: PMC3880289  PMID: 24404154
6.  Characterization of the Mouse Beta Defensin 1, Defb1, Mutant Mouse Model  
Infection and Immunity  2002;70(6):3053-3060.
Beta defensins are small cationic antimicrobial peptides present in the respiratory system which have been proposed to be dysfunctional in the environment of the cystic fibrosis lung. Defb1, a murine homologue to the human beta defensins, has also been found to be expressed in the respiratory system and, in order to examine the function of beta defensins in vivo, gene targeting was used to generate Defb1-deficient (Defb1tm1Hgu/Defb1tm1Hgu [Defb1−/−]) mice. The Defb1 synthetic peptide was shown to have a salt-sensitive antimicrobial activity that was stronger against Staphylococcus aureus than against Escherichia coli or Pseudomonas aeruginosa. Defb1−/− mice were found, however, to be effective in the clearance of the cystic fibrosis relevant pathogen S. aureus from the airways after nebulization. Although no overt deleterious phenotype was evident in the Defb1−/− mice, the number of mutant mice found to harbor bacteria of the Staphylococcus species in the bladder was significantly higher (P = 0.008) than that of controls, suggesting a role for these peptides in resistance to urinary tract infection.
doi:10.1128/IAI.70.6.3053-3060.2002
PMCID: PMC128030  PMID: 12010997
7.  Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus) 
Background
beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation.
Methods
In silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118–123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities.
Results
Novel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10–60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity.
Conclusion
Rat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis.
doi:10.1186/1477-7827-4-7
PMCID: PMC1420305  PMID: 16457734
8.  Narrowing down the distal border of the copy number variable beta-defensin gene cluster on human 8p23 
BMC Research Notes  2014;7:93.
Background
Copy number variation (CNV) in the range from 2 to 12 per diploid genome is an outstanding feature of the beta-defensin gene (DEFB) cluster on human chromosome 8p23.1 numerously demonstrated by different methods. So far, CNV was proven for a 115 kb region between DEFB4 and 21 kb proximal of DEFB107 but the borders for the entire CNV repeat unit are still unknown. Our study aimed to narrow down the distal border of the DEFB cluster.
Results
We established tests for length polymorphisms based on amplification and capillary electrophoresis with laser-induced fluorescence (CE-LIF) analysis of seven insertion/deletion (indel) containing regions spread over the entire cluster. The tests were carried out with 25 genomic DNAs with different previously determined cluster copy numbers. CNV was demonstrated for six indels between ~1 kb distal of DEFB108P and 10 kb proximal of DEFB107. In contrast, the most distal indel is not affected by CNV.
Conclusion
Our analysis fixes the minimal length of proven CNV to 157 kb including DEFB108P but excluding DEFB109P. The distal border between CNV and non-CNV part of the DEF cluster is located in the 59 kb interval chr8:7,171,082-7,230,128.
doi:10.1186/1756-0500-7-93
PMCID: PMC3942070  PMID: 24552181
Defensin; CNV borders; Length polymorphism; Paralog ratio test; Indel; DEFB108P; DEFB109P
9.  Copy Number Variation within Human β-Defensin Gene Cluster Influences Progression to AIDS in the Multicenter AIDS Cohort Study 
Study background
DEFB4/103A encoding β-defensin 2 and 3, respectively, inhibit CXCR4-tropic (X4) viruses in vitro. We determined whether DEFB4/103A Copy Number Variation (CNV) influences time-to-X4 and time-to-AIDS outcomes.
Methods
We utilized samples from a previously published Multicenter AIDS Cohort Study (MACS), which provides longitudinal account of viral tropism in relation to the full spectrum of rates of disease progression. Using traditional models for time-to-event analysis, we investigated association between DEFB4/103A CNV and the two outcomes, and interaction between DEFB4/103A CNV and disease progression groups, Fast and Slow.
Results
Time-to-X4 and time-to-AIDS were weakly correlated. There was a stronger relationship between these two outcomes for the fast progressors. DEFB4/103A CNV was associated with time-to-AIDS, but not time-to-X4. The association between higher DEFB4/103A CNV and time-to-AIDS was more pronounced for the slow progressors.
Conclusion
DEFB4/103A CNV was associated with time-to-AIDS in a disease progression group-specific manner in the MACS cohort. Our findings may contribute to enhancing current understanding of how genetic predisposition influences AIDS progression.
doi:10.4172/2155-6113.1000184
PMCID: PMC3610425  PMID: 23543857
AIDS progression; β-Defensin; DEFB4; DEFB103A; MACS; X4 emergence
10.  Increased Expression of Beta-Defensin 1 (DEFB1) in Chronic Obstructive Pulmonary Disease 
PLoS ONE  2011;6(7):e21898.
On-going airway inflammation is characteristic for the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the key factors determining the decrease in lung function, an important clinical parameter of COPD, are not clear. Genome-wide linkage analyses provide evidence for significant linkage to airway obstruction susceptibility loci on chromosome 8p23, the location of the human defensin gene cluster. Moreover, a genetic variation in the defensin beta 1 (DEFB1) gene was found to be associated with COPD. Therefore, we hypothesized that DEFB1 is differently regulated and expressed in human lungs during COPD progression. Gene expression of DEFB1 was assessed in bronchial epithelium and BAL fluid cells of healthy controls and patients with COPD and using bisulfite sequencing and ChIP analysis, the epigenetic control of DEFB1 mRNA expression was investigated. We can demonstrate that DEFB1 mRNA expression was significantly increased in bronchopulmonary specimen of patients with COPD (n = 34) vs. healthy controls (n = 10) (p<0.0001). Furthermore, a significant correlation could be detected between DEFB1 and functional parameters such as FEV1 (p = 0.0024) and the FEV1/VC ratio (p = 0.0005). Upregulation of DEFB1 mRNA was paralleled by changes in HDAC1-3, HDAC5 and HDAC8 mRNA expression. Whereas bisulfite sequencing revealed no differences in the methylation state of DEFB1 promoter between patients with COPD and controls, ChIP analysis showed that enhanced DEFB1 mRNA expression was associated with the establishment of an active histone code. Thus, expression of human DEFB1 is upregulated and related to the decrease in pulmonary function in patients with COPD.
doi:10.1371/journal.pone.0021898
PMCID: PMC3139569  PMID: 21818276
11.  Association studies of the copy-number variable ß-defensin cluster on 8p23.1 in adenocarcinoma and chronic pancreatitis 
BMC Research Notes  2012;5:629.
Background
Human ß-defensins are a family of antimicrobial peptides located at the mucosal surface. Both sequence multi-site variations (MSV) and copy-number variants (CNV) of the defensin-encoding genes are associated with increased risk for various diseases, including cancer and inflammatory conditions such as psoriasis and acute pancreatitis. In a case–control study, we investigated the association between MSV in DEFB104 as well as defensin gene (DEF) cluster copy number (CN), and pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP).
Results
Two groups of PDAC (N=70) and CP (N=60) patients were compared to matched healthy control groups CARLA1 (N=232) and CARLA2 (N=160), respectively. Four DEFB104 MSV were haplotyped by PCR, cloning and sequencing. DEF cluster CN was determined by multiplex ligation-dependent probe amplification.
Neither the PDAC nor the CP cohorts show significant differences in the DEFB104 haplotype distribution compared to the respective control groups CARLA1 and CARLA2, respectively.
The diploid DEF cluster CN exhibit a significantly different distribution between PDAC and CARLA1 (Fisher’s exact test P=0.027), but not between CP and CARLA2 (P=0.867).
Conclusion
Different DEF cluster b CN distribution between PDAC patients and healthy controls indicate a potential protective effect of higher CNs against the disease.
doi:10.1186/1756-0500-5-629
PMCID: PMC3532138  PMID: 23148552
Defensins; Single nucleotide variants; Copy number variation; Chronic pancreatitis; Pancreatic ductal adenocarcinoma
12.  β-Defensin Genomic Copy Number Does Not Influence the Age of Onset in Huntington’s Disease 
Journal of Huntington's disease  2013;2(1):107-124.
Background
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of a CAG triplet repeat tract in the huntingtin gene. While the length of this CAG expansion is the major determinant of the age of onset (AO), other genetic factors have also been shown to play a modulatory role. Recent evidence suggests that neuroinflammation is a pivotal factor in the pathogenesis of HD, and that targeting this process may have important therapeutic ramifications. The human β-defensin 2 (hBD2) – encoded by DEFB4 – is an antimicrobial peptide that exhibits inducible expression in astrocytes during inflammation and is an important regulator of innate and adaptive immune response. Therefore, DEFB4 may contribute to the neuroinflammatory processes observed in HD.
Objective
In this study we tested the hypothesis that copy number variation (CNV) of the β-defensin region, including DEFB4, modifies the AO in HD.
Methods and results
We genotyped β-defensin CNV in 490 HD individuals using the paralogue ratio test and found no association between β-defensin CNV and onset of HD.
Conclusions
We conclude that it is unlikely that DEFB4 plays a role in HD pathogenesis.
doi:10.3233/JHD-130047
PMCID: PMC3935171  PMID: 24587836
Genetic modifier; copy number variation; inflammation
13.  Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing 
BMC Genomics  2010;11:252.
Background
The beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel.
Results
Six PCR products spread over ~87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From ~142,000 reads, ~120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed.
Conclusion
Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.
doi:10.1186/1471-2164-11-252
PMCID: PMC2873476  PMID: 20403190
14.  A genome-wide association study of variants associated with acquisition of Staphylococcus aureus bacteremia in a healthcare setting 
Background
Humans vary in their susceptibility to acquiring Staphylococcus aureus infection, and research suggests that there is a genetic basis for this variability. Several recent genome-wide association studies (GWAS) have identified variants that may affect susceptibility to infectious diseases, demonstrating the potential value of GWAS in this arena.
Methods
We conducted a GWAS to identify common variants associated with acquisition of S. aureus bacteremia (SAB) resulting from healthcare contact. We performed a logistic regression analysis to compare patients with healthcare contact who developed SAB (361 cases) to patients with healthcare contact in the same hospital who did not develop SAB (699 controls), testing 542,410 SNPs and adjusting for age (by decade), sex, and 6 significant principal components from our EIGENSTRAT analysis. Additionally, we evaluated the joint effect of the host and pathogen genomes in association with severity of SAB infection via logistic regression, including an interaction of host SNP with bacterial genotype, and adjusting for age (by decade), sex, the 6 significant principal components, and dialysis status. Bonferroni corrections were applied in both analyses to control for multiple comparisons.
Results
Ours is the first study that has attempted to evaluate the entire human genome for variants potentially involved in the acquisition or severity of SAB. Although this study identified no common variant of large effect size to have genome-wide significance for association with either the risk of acquiring SAB or severity of SAB, the variant (rs2043436) most significantly associated with severity of infection is located in a biologically plausible candidate gene (CDON, a member of the immunoglobulin family) and may warrant further study.
Conclusions
The genetic architecture underlying SAB is likely to be complex. Future investigations using larger samples, narrowed phenotypes, and advances in both genotyping and analytical methodologies will be important tools for identifying causative variants for this common and serious cause of healthcare-associated infection.
doi:10.1186/1471-2334-14-83
PMCID: PMC3928605  PMID: 24524581
Genomics; Genome-wide association study; Case–control study; Staphylococcus aureus; Bacteremia; Gram-positive bacterial infections; Polymorphism, single-nucleotide; Infections; Nosocomial; Cross infection
15.  Association of Mannose-Binding Lectin 2 Gene Polymorphisms with Persistent Staphylococcus aureus Bacteremia 
PLoS ONE  2014;9(3):e89139.
Objectives
Mannose-binding lectin (MBL) is an important component of innate immunity. Structural and promoter polymorphisms in the MBL2 gene that are responsible for low MBL levels are associated with susceptibility to infectious diseases. The objective of this study was to investigate the association of serum MBL levels and MBL2 polymorphisms with persistent Staphylococcus aureus bacteremia (SAB) in adult Korean patients.
Methods
We conducted a case-control study nested in a prospective cohort of patients with SAB. The study compared 41 patients with persistent bacteremia (≥7 days) and 46 patients with resolving bacteremia (<3 days). In each subject, we genotyped six single-nucleotide polymorphisms in the promoter region (alleles H/L, X/Y, and P/Q) and exon 1 (alleles A/B, A/C, and A/D) of the MBL2 gene and measured serum MBL concentrations. We also compared MBL2 genotypes between SAB patients and healthy people.
Results
Patients with persistent bacteremia were significantly more likely to have low/deficient MBL-producing genotypes and resultant low serum MBL levels, than were patients with resolving bacteremia (P = 0.019 and P = 0.012, respectively). Independent risk factors for persistent bacteremia were metastatic infection (adjusted odds ratio [aOR], 34.7; 95% confidence interval [CI], 12.83–196.37; P = 0.003), methicillin resistance (aOR, 4.10; 95% CI, 3.19–29.57; P = 0.025), and low/deficient MBL-producing genotypes (aOR, 7.64; 95% CI, 4.12–63.39; P = 0.003). Such genotypes were significantly more common in patients with persistent bacteremia than in healthy people (OR, 2.09; 95% CI, 1.03–4.26; P = 0.040).
Conclusions
This is the first demonstration of an association of low MBL levels and MBL2 polymorphisms responsible for low or deficient MBL levels with persistent SAB. A combination of factors, including clinical and microbiological characteristics and host defense factors such as MBL levels, may together contribute to the development of persistent SAB.
doi:10.1371/journal.pone.0089139
PMCID: PMC3942407  PMID: 24595015
16.  Outcome of Vancomycin Treatment in Patients with Methicillin-Susceptible Staphylococcus aureus Bacteremia▿  
Limited data on the clinical outcome of vancomycin treatment compared with that of beta-lactam treatment in patients with methicillin-susceptible Staphylococcus aureus bacteremia (MSSA-B) are available. We used different and complementary approaches: (i) a retrospective cohort study using a propensity score to adjust for confounding by treatment assignment and (ii) a matched case-control study. Of all patients with S. aureus bacteremia (SAB) in two university-affiliated hospitals over a 7-year period, 294 patients with MSSA-B were enrolled in the cohort study. The cases for the case-control study were defined as patients who received vancomycin treatment for MSSA-B; the controls, who were patients that received beta-lactam treatment for MSSA-B, were selected at a 1:2 (case:control) ratio according to the objective matching scoring system and the propensity score system. In the cohort study, SAB-related mortality in patients with vancomycin treatment (37%, 10/27) was significantly higher than that in those with beta-lactam treatment (18%, 47/267) (P = 0.02). In addition, multivariate analysis revealed that vancomycin treatment was associated with SAB-related mortality when independent predictors for SAB-related mortality and propensity score were considered (adjusted odds ratio of 3.3, 95% confidence interval of 1.2 to 9.5). In the case-control study using the objective matching scoring system and the propensity score system, SAB-related mortality in case patients was 37% (10/27) and in control patients 11% (6/54) (P < 0.01). Our data suggest that vancomycin is inferior to beta-lactam in the treatment of MSSA-B.
doi:10.1128/AAC.00700-07
PMCID: PMC2223910  PMID: 17984229
17.  Histone Deacetylase (HDAC) 1 Controls the Expression of Beta Defensin 1 in Human Lung Epithelial Cells 
PLoS ONE  2012;7(11):e50000.
Deregulation of the expression human beta defensin 1 (DEFB1), an antimicrobial peptide, has been implicated in the pathogenesis of COPD and asthma. Since the molecular mechanisms that regulate DEFB1 gene expression are widely unknown, the epigenetic processes involved in the regulation of the constitutive expression of DEFB1 in lung epithelial cells (A549) were investigated. The data demonstrate that histone deacetylases (HDACs) participate in the regulation of DEFB1 gene expression. Inhibition of the class I HDACs, HDACs 1-3, increases DEFB1 gene expression in A549 cells. Chromatin immunoprecipitation (ChIP) assays revealed that the inhibition of the class I HDACs also results in modifications of the chromatin at the DEFB1 promoter. Histone modifications, histone H3 acetylation and H3K4 trimethylation, that are associated with transcriptional activation, were found to increase after inhibition of HDACs 1-3. Finally, RNAi knockdown experiments identified HDAC1 as the sole HDAC responsible for maintaining the constitutive level of DEFB1 transcription. Taken together, our data reveal epigenetic mechanisms which are the basis of the maintenance of the constitutive gene expression of human beta defensin 1.
doi:10.1371/journal.pone.0050000
PMCID: PMC3502185  PMID: 23185513
18.  Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats 
Nucleic Acids Research  2006;35(3):e19.
Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.
doi:10.1093/nar/gkl1089
PMCID: PMC1807953  PMID: 17175532
19.  Temporal Trends in Incidence of Staphylococcus aureus Bacteremia in Olmsted County, Minnesota, 1998 to 2005: A Population-Based Study 
Background
There is a paucity of population-based studies on Staphylococcus aureus bacteremia (SAB) in the United States. We determined the incidence and trends of SAB in Olmsted County, Minnesota, over an 8-year period.
Methods
A retrospective, population-based, cohort study was done to evaluate the initial episodes of SAB occurring in adult residents of Olmsted County, Minnesota, from January 1, 1998 through December 31, 2005 using the microbiology databases at Mayo Clinic and Olmsted Medical Center.
Results
Among 247 evaluable adult patients with SAB, who were included in the incidence calculation, 57.9% were males and the median age was 72.1 years (range 19.5 - 98.5). Bacteremic episodes were classified according to acquisition site: 23.5% were nosocomial (NA-SAB); 58.7% were healthcare-associated (HCA-SAB); and 23.8% were community-acquired (CA-SAB). MRSA constituted 31.6% of the cases. No community-acquired MRSA bacteremia was noted. The age-adjusted incidence rate of SAB was 28.3/100,000 person-years for females, and 53.5/100,000 person-years for males, with an age- and gender-adjusted rate of 38.2/100,000 person-years. The age- and gender-adjusted incidence of NA-SAB, HCA-SAB, and CA-SAB was 9.0, 22.6 and 6.6/100,000 person-years, respectively. The age- and gender-adjusted incidence of MSSA was 25.4/100,000 and of MRSA was 12.4/100,000 person-years. Overall, the incidence rate increased with age, but not over calendar year. The exception was MRSA-B, which increased at a rate of 19.8% (± 5.5%) per year during the study period.
Conclusions
The incidence of SAB in adults remained stable in Olmsted County, Minnesota, from 1998 to 2005, but the proportion due to MRSA has significantly increased over the 8-year period.
doi:10.1086/648442
PMCID: PMC3050712  PMID: 19916797
Incidence; Staphylococcus aureus; bacteremia; population-based; Olmsted County
20.  Gastric Antimicrobial Peptides Fail to Eradicate Helicobacter pylori Infection Due to Selective Induction and Resistance 
PLoS ONE  2013;8(9):e73867.
Background
Although antimicrobial peptides protect mucus and mucosa from bacteria, Helicobacter pylori is able to colonize the gastric mucus. To clarify in which extend Helicobacter escapes the antimicrobial defense, we systematically assessed susceptibility and expression levels of different antimicrobial host factors in gastric mucosa with and without H. pylori infection.
Materials and Methods
We investigated the expression levels of HBD1 (gene name DEFB1), HBD2 (DEFB4A), HBD3 (DEFB103A), HBD4 (DEFB104A), LL37 (CAMP) and elafin (PI3) by real time PCR in gastric biopsy samples in a total of 20 controls versus 12 patients colonized with H. pylori. Immunostaining was performed for HBD2 and HBD3. We assessed antimicrobial susceptibility by flow cytometry, growth on blood agar, radial diffusion assay and electron microscopy.
Results
H. pylori infection was associated with increased gastric levels of the inducible defensin HBD2 and of the antiprotease elafin, whereas the expression levels of the constitutive defensin HBD1, inducible HBD3 and LL37 remained unchanged. HBD4 was not expressed in significant levels in gastric mucosa. H. pylori strains were resistant to the defensins HBD1 as well as to elafin, and strain specific minimally susceptible to HBD2, whereas HBD3 and LL37 killed all H. pylori strains effectively. We demonstrated the binding of HBD2 and LL37 on the surface of H. pylori cells. Comparing the antibacterial activity of extracts from H. pylori negative and positive biopsies, we found only a minimal killing against H. pylori that was not increased by the induction of HBD2 in H. pylori positive samples.
Conclusion
These data support the hypothesis that gastric H. pylori evades the host defense shield to allow colonization.
doi:10.1371/journal.pone.0073867
PMCID: PMC3770654  PMID: 24040100
21.  Clinical application of human β-defensin and CD14 gene polymorphism in evaluating the status of chronic inflammation 
Journal of Translational Medicine  2012;10(Suppl 1):S9.
Background
Periodontitis is a common disease that affects the periodontal tissue supporting the teeth. This disease is attributed to multiple risk factors, including diabetes, cigarette smoking, alcohol, pathogenic microorganisms, genetics and others. Human beta-defensin-1 (hBD-1) is a cationic antimicrobial peptide with cysteine-rich ß-sheets and broad-spectrum antimicrobial activity. CD14 is a protein involved in the detection of bacterial lipopolysaccharide (LPS) and has also been associated with periodontitis. This study investigates the single nucleotide polymorphic (SNP) region, -1654(V38I), of the human beta-defensin-1 (hBD-1) gene as well as the -159 region of the CD14 gene in subjects with chronic periodontitis.
Methods
Blood samples from periodontally healthy subjects and periodontitis patients were obtained. DNA was extracted from the blood and was used to perform restriction digest at the polymorphic G1654A site of DEFB1 with the enzyme HincII. The polymorphic site 159TT of CD14 was digested with the enzyme AvaII. Enzyme-linked immunosorbent assay (ELISA) was performed on soluble samples to determine the protein expressions.
Results
The control and patient groups expressed 35% and 38% 1654 A/A genotype of DEFB1, respectively. The A allele frequency of the control group was 40%, while the patient blood group was 54%. The mean hBD-1 protein levels of the control and patient samples were 102.83 pg/mL and 252.09 pg/mL, respectively. The genotype distribution of CD14 in healthy subjects was 16% for C/C, 26% for T/T and 58% for C/T. The genotype frequencies of CD14 in periodontitis patients were 10% for C/C, 43% for T/T and 47% for C/T. The CD14 protein expression determined by ELISA showed a mean protein level of the control samples at 76.28ng/mL and the patient blood samples at 179.27ng/mL with a p value of 0.001.
Our study demonstrated that patients suffering from chronic periodontitis present more commonly with the 1654A/A genotype on the DEFB1 gene and the 159T/T genotype on the CD14 gene.
Conclusions
This study purely investigated the association between periodontitis and one polymorphic site on both DEFB1 and CD14 gene, with the purpose of expanding knowledge for the future development in diagnostic markers or therapeutic interventions to combat this disease.
doi:10.1186/1479-5876-10-S1-S9
PMCID: PMC3445860  PMID: 23046822
22.  Androgenic regulation of beta-defensins in the mouse epididymis 
Background
The majority of beta-defensin family members are exclusively expressed in the epididymis, and some members have been shown to play essential roles in sperm maturation and fertility in rats, mice and humans. Therefore, beta-defensins are hypothesized to be potential targets for contraception and infertility diagnosis and treatment. Clarifying the regulatory mechanisms for the expression of these genes is necessary. Androgen/androgen receptor (AR) signaling plays an important regulatory role in epididymal structure and function. However, very little is known about the androgenic regulation on the production and secretion of the epididymal beta-defensins.
Methods
The expression of beta-defensins was detected by quantitative RT-PCR. The androgen dependence of beta-defensins was determined by bilateral orchiectomy and androgen supplementation. The androgen response elements (AREs) in the promoters of beta-defensins were identified using the MatInspector software. The binding of AR to AREs was assayed by ChIP-PCR/qPCR.
Results
We demonstrated that 23 mouse caput epididymal beta-defensins were differentially regulated by androgen/androgen receptor. Six genes, Defb18, 19, 20, 39, 41, and 42, showed full regulation by androgens. Ten genes, Defb15, 30, 34, 37, 40, 45, 51, 52, 22 and Spag11a, were partially regulated by androgens. Defb15, 18, 19, 20, 30, 34, 37, 39, 41, 42, 22 and Spag11a were associated with androgen receptor binding sites in their promoter or intronic regions, indicating direct regulation of AR. Six genes, Defb1, 12, 13, 29, 35, and spag11b/c, exhibited an androgen-independent expression pattern. One gene, Defb25, was highly dependent on testicular factors rather on androgens.
Conclusions
The present study provides novel insights into the mechanisms of androgen regulation on epididymal beta-defensins, enabling a better understanding of the function of beta-defensins in sperm maturation and fertility.
doi:10.1186/1477-7827-12-76
PMCID: PMC4127520  PMID: 25099571
Androgen; Androgen receptor; Epididymis; Beta-defensins
23.  Increased Levels of Human Beta-Defensins mRNA in Sexually HIV-1 Exposed But Uninfected Individuals 
Current HIV research  2008;6(6):531-538.
Protection against HIV-1 infection in exposed seronegative (ESN) individuals likely involves natural resistance mechanisms that have not been fully elucidated. Human beta defensins (HBD) are antimicrobial peptides found primarily in mucosae, the main ports of HIV entry. HBD-2 and 3 mRNA are induced by HIV-1 in human oral epithelial cells and exhibit strong anti-HIV-1 activity; in addition, polymorphisms in the DEFB1 gene, which encodes HBD-1, have been associated with resistance/susceptibility to different infections, including HIV-1. Here, we have assessed the association of HBD expression with the ESN phenotype. Peripheral blood and vaginal/endocervical and oral mucosal samples were taken from 47 ESN, 44 seropositive (SP) and 39 healthy controls (HC). HBD-1, 2 and 3 mRNA copy numbers were quantified by real time RT-PCR and A692G/G1654A/A1836G polymorphisms in the DEFB1 gene were detected by restriction fragment length polymorphisms and confirmed by nucleotide sequencing. ESN expressed significantly greater mRNA copy numbers of HBD-2 and 3 in oral mucosa than HC; p=0.0002 and p=0.007, respectively. mRNA copy numbers of HBD-1, 2 and 3 in vaginal/endocervical mucosa from ESN and HC were similar. Homozygosity for the A692G polymorphism was significantly more frequent in ESN (0.39) than in SP (0.05) (p=0.0002). In summary, ESN exhibited enhanced mucosal expression of the innate defense genes HBD-2 and 3; however, additional studies are required to verify these results and the potential association of the A692G polymorphism to the relative resistance of ESN to HIV-1 infection.
PMCID: PMC4126611  PMID: 18991618
HIV-1 (Human immunodeficiency virus type 1); human beta defensins; natural resistance; HIV-1-exposed sero-negatives; polymorphism; mucosa
24.  A Worldwide Analysis of Beta-Defensin Copy Number Variation Suggests Recent Selection of a High-Expressing DEFB103 Gene Copy in East Asia 
Human Mutation  2011;32(7):743-750.
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. Hum Mutat 32:743–750, 2011. © 2011 Wiley-Liss, Inc.
doi:10.1002/humu.21491
PMCID: PMC3263423  PMID: 21387465
CNV; defensin; antimicrobial; influenza; paralogue ratio test
25.  Stable incidence and continued improvement in short term mortality of Staphylococcus aureus bacteraemia between 1995 and 2008 
BMC Infectious Diseases  2012;12:260.
Background
The objective of this study was to assess temporal changes in incidence and short term mortality of Staphylococcus aureus bacteraemia (SAB) from 1995 through 2008.
Methods
The study was conducted as a nation-wide observational cohort study with matched population controls. The setting was hospitalized patients in Denmark 1995-2008. Uni- and multivariate analyses were used to analyze the hazard of death within 30 days from SAB.
Results
A total of 16 330 cases of SAB were identified: 57% were hospital-associated (HA), 31% were community-acquired (CA) and 13% were of undetermined acquisition. The overall adjusted incidence rate remained stable at 23 per 100 000 population but the proportion of SAB cases older than 75 years increased significantly. Comorbidity in the cohort as measured by Charlson comorbidity index (CCI) score and alcohol-related diagnoses increased over the study period. In contrast, among the population controls the CCI remained stable and alcohol-related diagnoses increased slightly. For HA SAB crude 30-day mortality decreased from 27.8% to 21.8% (22% reduction) whereas the change for CA SAB was small (26.5% to 25.8%). By multivariate Cox regression, age, female sex, time period, CCI score and alcohol-related diagnoses were associated with increased mortality regardless of mode of acquisition.
Conclusions
Throughout a 14-year period the overall incidence of SAB remained stable while the overall short term prognosis continued to improve despite increased age and accumulation of comorbidity in the cohort. However, age and comorbidity were strong prognostic indicators for short term mortality.
doi:10.1186/1471-2334-12-260
PMCID: PMC3507819  PMID: 23075215
Bacteraemia; Epidemiology; Incidence; Mortality; Comorbidity; Alcoholism; Staphylococcus aureus; Charlson comorbidity index

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