Phenotypic Consequences of β-Defensin Copy Number Variation in Humans

Beta defensins (DEFB) at the 8p23.1 genomic location are multifunctional secreted short peptides that have antibacterial and antiviral action in many species and possess immune cell signal activity, constituting a link between innate and adaptive immunity. In humans, the β-defensin region is known to be copy number variable (CNV) and contains seven genes repeated as a block, with a diploid copy number between 1 and 12. This thesis shall explore the structural variability of the β-defensin CNV region; compare and contrast the different methods used for calling DEFB CNVs and investigate the role of CNVs of DEFB in various diseases. One of its aims is to also develop a model system to investigate if DEFB expression levels differ with CN in response to treatment with Pneumolysin by using Normal Human Bronchial Epithelial (NHBE) cells. Results from this thesis confirm that the DEFB CNV region is 322kb in length, with a polymorphic inversion that occurs at a prevalence of 30% at the 8p23.1 genomic location that is independent of the DEFB CN. Paralogue Ratio Test (PRT) proved to be the best method of genotyping DEFB CNV especially in larger cohorts. In addition, work from this thesis also founded the basis of developing an in vitro model system to investigate whether DEFB expression levels differ with CN in response to treatment with pneumolysin by using Normal Human Bronchial Epithelial (NHBE) cells. As far as case/control and cohort studies are concerned, results from this thesis show that DEFB CN is not associated with lung function in the general population and has no effect on patients with COPD and Asthma, nor does it support previous results that present an association between HIV viral load and DEFB CN. DEFB CN was also found not to be associated with recurrent UTIs in VUR patients, nor with hypertension. Data suggested that DEFB CN might be associated with BMI but this has not been reproduced in a smaller cohort

Additional file 1: Table S1. of Determining multiallelic complex copy number and sequence variation from high coverage exome sequencing data

Frequency of rs200757797 and rs2740090 in the 1000 Genomes continental groups. (DOCX 13.4 kb

Determining multiallelic complex copy number and sequence variation from high coverage exome sequencing data

BACKGROUND: Copy number variation (CNV) is a major component of genomic variation, yet methods to accurately type genomic CNV lag behind methods that type single nucleotide variation. High-throughput sequencing can contribute to these methods by using sequence read depth, which takes the number of reads that map to a given part of the reference genome as a proxy for copy number of that region, and compares across samples. Furthermore, high-throughput sequencing also provides information on the sequence differences between copies within and between individuals. METHODS: In this study we use high-coverage phase 3 exome sequences of the 1000 Genomes project to infer diploid copy number of the beta-defensin genomic region, a well-studied CNV that carries several beta-defensin genes involved in the antimicrobial response, signalling, and fertility. We also use these data to call sequence variants, a particular challenge given the multicopy nature of the region. RESULTS: We confidently call copy number and sequence variation of the beta-defensin genes on 1285 samples from 26 global populations, validate copy number using Nanostring nCounter and triplex paralogue ratio test data. We use the copy number calls to verify the genomic extent of the CNV and validate sequence calls using analysis of cloned PCR products. We identify novel variation, mostly individually rare, predicted to alter amino-acid sequence in the beta-defensin genes. Such novel variants may alter antimicrobial properties or have off-target receptor interactions, and may contribute to individuality in immunological response and fertility. CONCLUSIONS: Given that 81 % of identified sequence variants were not previously in dbSNP, we show that sequence variation in multiallelic CNVs represent an unappreciated source of genomic diversity

Human beta defensin (HBD) gene copy number affects HBD2 protein levels: impact on cervical bactericidal immunity in pregnancy

Human beta defensin 2 (HBD2) is an endogenous mucosal antimicrobial peptide (AMP) upregulated during infection and inflammation. HBD2 is encoded by the DEFB4 gene, which exhibits extensive copy number variation. Previous studies have demonstrated a relationship between HBD copy number and serum HBD2 protein levels; however, our current understanding of the influence of copy number on mucosal AMP function remains limited. This study explores the relationship between HBD copy number, cervicovaginal HBD2 protein levels and antimicrobial activity in 203 women with risk factors for preterm birth. We provide evidence that suggests HBD copy number modulates cervical antimicrobial immunity

No Evidence for Association of β-Defensin Genomic Copy Number with HIV Susceptibility, HIV Load during Clinical Latency, or Progression to AIDS

Common single nucleotide variation in the host accounts for 25% of the variability in the plasma levels of HIV during the clinical latency stage (viral load setpoint). However, the role of rare variants and copy number variants remains relatively unexplored. Previous work has suggested copy number variation of a cluster of β-defensin genes affects HIV load in treatment-naïve sub-Saharan Africans and rate of response to anti-retroviral treatment. Here we analyse a total of 1827 individuals from two cohorts of HIV-infected individuals from Europe and sub-Saharan Africa to investigate the role of β-defensin copy number variation on HIV load at setpoint. We find no evidence of for association of copy number with viral load. We also compare distribution of β-defensin copy number between European cases and controls and find no differences, arguing against a role of β-defensin copy number in HIV acquisition. Taken together, our data argues against an effect of copy number variation of the β-defensin region in the spontaneous control of HIV infection

Evidence of convergent evolution in humans and macaques supports an adaptive role for copy number variation of the β-defensin-2 gene.

β-defensins are a family of important peptides of innate immunity, involved in host defense, immunomodulation, reproduction, and pigmentation. Genes encoding β-defensins show evidence of birth-and-death evolution, adaptation by amino acid sequence changes, and extensive copy number variation (CNV) within humans and other species. The role of CNV in the adaptation of β-defensins to new functions remains unclear, as does the adaptive role of CNV in general. Here, we fine-map CNV of a cluster of β-defensins in humans and rhesus macaques. Remarkably, we found that the structure of the CNV is different between primates, with distinct mutational origins and CNV boundaries defined by retroviral long terminal repeat elements. Although the human β-defensin CNV region is 322 kb and encompasses several genes, including β-defensins, a long noncoding RNA gene, and testes-specific zinc-finger transcription factors, the orthologous region in the rhesus macaque shows CNV of a 20-kb region, containing only a single gene, the ortholog of the human β-defensin-2 gene. Despite its independent origins, the range of gene copy numbers in the rhesus macaque is similar to humans. In addition, the rhesus macaque gene has been subject to divergent positive selection at the amino acid level following its initial duplication event between 3 and 9.5 Ma, suggesting adaptation of this gene as the macaque successfully colonized novel environments outside Africa. Therefore, the molecular phenotype of β-defensin-2 CNV has undergone convergent evolution, and this gene shows evidence of adaptation at the amino acid level in rhesus macaques

DEFB copy number in LRC asthma-ICS cases and controls.

<p>DEFB copy number in LRC asthma-ICS cases and controls.</p

Characteristics of Gedling and replication COPD cases and controls.

<p>Characteristics of Gedling and replication COPD cases and controls.</p

Distributions of raw PRT copy number (top) and maximum likelihod integer copy number (bottom) for Gedling (right) and LRC (left) cohorts.

<p>Distributions of raw PRT copy number (top) and maximum likelihod integer copy number (bottom) for Gedling (right) and LRC (left) cohorts.</p

Sample selection for association analyses in Gedling and LRC.

<p>The sampling frame for each cohort is shown centrally with the COPD and asthma association analysis sample subsets indicated above and below for each cohort.</p