Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Boniotto M

PROVIDER: S-EPMC440632 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Boniotto Michele M Hazbón Manzour Hernando MH Jordan William James WJ Lennon Greig Patrick GP Eskdale Joyce J Alland David D Gallagher Grant G

Clinical and diagnostic laboratory immunology 20040701 4

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 ...[more]

PMID: 15242954

Similar Datasets

Project description:Human beta-defensins (hBDs) are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP) in the 5' untranslated region of the hBD1 gene (DEFB1), denoted -44 (rs1800972), is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression.Transfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression.Transfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1beta, TNFalpha, and interferon-gamma (IFNgamma). In contrast to constitutive expression levels, IFNgamma-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression.The DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNgamma inducibility of these beta-defensins and is likely to be more protective in conditions that enhance IFNgamma expression such as chronic periodontitis. These results suggest a complex relationship between genetics and defensin expression that may influence periodontal health and innate immune responses.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets