Correlation of salivary β-defensin peptide levels with defensin copy numbers

The defensin family of peptides represents epithelial expressed cationic antimicrobial agents that serve as protective components of the epithelial mucosal barrier. Genetic variation of the defensin genes in the form of single nucleotide polymorphism (SNPs) and copy number variants (CNVs) have been shown to be associated with disease susceptibility. The defensin gene cluster is mapped to chromosome 8p22-23p with DEFB1 encoding the constitutively expressed hBD-1 peptide and DEFB4 and 103A encoding inducible peptides hBD-2 and hBD-3 respectively. DEFB1 SNPs (-52, -44, -20) have been shown to be responsible for an increase in peptide expression. DEFB1 has 2 copies per diploid genome (PDG) whereas the DEFB4 and DEFB103A genes have a range of 2-12 copies PDG. Objectives: To test if increased defensin salivary peptide expression level are related to both specific haplotype in DEFB1 and increased copy number in DEFB4 and DEFB103A. Methods: We evaluated ethnically diverse healthy individuals for salivary defensin peptide levels and genotyped them for SNPs in DEFB1 and CNVs in DEFB4 and DEFB103A. High throughput SNP assays were developed using a multiplex ligase detection reaction assay (MLDR), for DEFB1 haplotype determination. Quantitative real time PCR assays (qPCR) were performed for copy number determination in the DEFB4 and DEFB103A genes. Unstimulated whole saliva was collected and quantification of hBD-1, 2 and 3 were performed using a modified sandwiched ELISA technique. Results: Copy number did not directly correlate with peptide level in this study of healthy individuals. Conclusions: The individuals enrolled in this study had normal non inflamed oral mucosa. Since DEFB4 and DEFB103 A are inducible, correlation may be revealed only during mucosal inflammation or microbial challenge. Supported by NIH/NIDCR 1 K23 DE016110-01A1; RO1DE016334; RO1DE018276