Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay.

Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.

Endoplasmic reticulum aminopeptidase 1 polymorphism Ile276Met is associated with atopic dermatitis and affects the generation of an HLA-C associated antigenic epitope in vitro.

BACKGROUND: Atopic dermatitis (AD) is a common inflammatory skin disease of complex aetiology, with interactions between susceptibility genes and environmental factors. We have previously described a protective effect of the KIR2DS1 gene encoding the natural killer cell receptor, whose ligands are HLA-C molecules. Here, we found an association of HLA-C*05:01 allele with AD. KIR-HLA-C interactions are affected by peptides presented by HLA-C. The generation of these peptides is strongly influenced by endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2). Expression and activity of ERAP molecules depend on the polymorphisms of their genes. OBJECTIVE: Possible associations of several single nucleotide polymorphisms (SNPs) in the ERAP1 and ERAP2 genes with susceptibility to AD. METHODS: Peripheral blood DNA isolation from 318 patients and 549 controls. PCR-SSO or PCR-SSP for HLA-C typing; TaqMan Genotyping Assay for ERAP typing. RESULTS: Only one SNP in the ERAP1 gene, rs26618T>C, causing the amino acid change Ile276Met, had an association with AD. To gain insight on the functional role of this SNP, we produced recombinant variants differing only at position 276 (Ile or Met) and tested their aminopeptidase activity against a N-terminally extended precursor LIVDRPVTLV of the HLA-C*05:01 epitope IVDRPVTLV. Both ERAP1 variants were able to efficiently generate the epitope, although the 276Ile allotype was able to do this about 50% faster. Furthermore, both variants were quite inefficient in further degradation of the mature epitope. Finally, we found that the effect of 276Met on susceptibility to AD was seen only in KIR2DS1-negative individuals, not protected by this KIR. CONCLUSION: Associations of HLA-C*05:01 allele and rs26618T>C (Ile276Met) ERAP1 polymorphism with AD, and a significant difference between these two ERAP1 variants in their ability to generate an epitope for the HLA-C*05:01 molecule was found.