RESUMO
BACKGROUND: In times of genotype guided therapy options, a total of 3.2 % of people with CF (pwCF) in the German CF Registry[1] only have one or no CFTR-variant detected after genetic analysis. Additionally, genetic data in the Registry can be documented as free text and can therefore be prone to error. In order to allow the greatest possible amount of pwCF access to modern therapies, we conducted a re-evaluation of free text entries and established a custom-whole-CFTR-locus NGS-approach for all pwCF who remained without genetic confirmation afterwards. METHODS: To this end, we assembled 731 free text variants of 655 pwCF in the German CF Registry. All variants were evaluated using ClinVar, HGMD and CFTR1/2, corrected in the Registries' database and uploaded to ClinVar. PwCF whose diagnosis remained uncertain as well as additional pwCF or pwCFTR-RD that were assembled through a nationwide call for testing of unclear cases were offered genetic analysis. Samples were analysed using a target-capture based NGS-custom-design-panel covering the entire CFTR-locus. RESULTS: Evaluation of free text variants led to the discovery of 43 variants not formerly reported in the context of CF. The Registries' dropdown list was extended by 497 variants and over 500 pwCF were provided with their most up-to-date genotype. Samples of 47 pwCF/pwCFTR-RD were sequenced via NGS with an overall success rate of 61.7 %, resulting in implementation of entire CFTR-genotyping into routine diagnostics. CONCLUSION: Entire CFTR-genotyping can greatly increase the genetic diagnostic rate of pwCF/pwCFTR-RD and should be considered after inconspicuous CFTR screening panels in CFTR-diagnostics.
RESUMO
DNA methylation is pivotal in orchestrating gene expression patterns in various mammalian biological processes. Perturbation of the bovine alveolar macrophage (bAM) transcriptome, due to Mycobacterium bovis (M. bovis) infection, has been well documented; however, the impact of this intracellular pathogen on the bAM epigenome has not been determined. Here, whole genome bisulfite sequencing (WGBS) was used to assess the effect of M. bovis infection on the bAM DNA methylome. The methylomes of bAM infected with M. bovis were compared to those of non-infected bAM 24 hours post-infection (hpi). No differences in DNA methylation (CpG or non-CpG) were observed. Analysis of DNA methylation at proximal promoter regions uncovered >250 genes harbouring intermediately methylated (IM) promoters (average methylation of 33-66%). Gene ontology analysis, focusing on genes with low, intermediate or highly methylated promoters, revealed that genes with IM promoters were enriched for immune-related GO categories; this enrichment was not observed for genes in the high or low methylation groups. Targeted analysis of genes in the IM category confirmed the WGBS observation. This study is the first in cattle examining genome-wide DNA methylation at single nucleotide resolution in an important bovine cellular host-pathogen interaction model, providing evidence for IM promoter methylation in bAM.