Weisenberger, D. J. | bioRxiv Science

bioRxiv2026

Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.

Genomic, transcriptomic and epigenomic characterization has accelerated the discovery of clinically-relevant alterations in cancer, predominantly using fresh frozen (FF) specimens. However, clinical molecular pathology laboratories prefer formalin-fixed paraffin-embedded (FFPE) methods, known to introduce artifacts at the nucleic acid level, over fresh frozen methods. Extending the multi-platform analysis to FFPE specimens for comprehensive clinical molecular diagnosis requires a thorough understanding of the consequence of formalin-fixation. We present a detailed multi-platform characterization of FFPE preservation using paired FF specimens as the 'gold standard'. DNA and RNA were obtained from 38 patients across 6 cancer types using a FFPE optimized co-isolation. The impact of FFPE on exome sequencing was dependent on filtering, where a minimum coverage or supporting read filter can mitigate FFPE-specific false positives. Copy number alterations, MSI assessment, mutational signatures, and DNA methylation were comparable between FFPE and FF. FFPE biases in RNA expression can be overcome when using biology-relevant genes and we describe a novel consequence of FFPE on miRNA species diversity. Collectively, this data provides a broad view of FFPE artifact and offers best practices for overcome these biases.

bioinformatics↗

bioRxiv2019

Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy

Homeostatic maintenance of corneal endothelial cells is essential for maintenance of corneal deturgescence and corneal transparency. In Fuchs endothelial corneal dystrophy (FECD), an accelerated loss and dysfunction of endothelial cells leads to progressively severe visual impairment. An abnormal accumulation of extracellular matrix is a distinctive hallmark of the disease, however the molecular pathogenic mechanisms underlying this phenomenon are not fully understood. We recently reported characteristic patterns of DNA methylation changes in the corneal endothelial cells of patients with FECD. Here, we investigate genome-wide and sequence-specific DNA methylation changes of miRNA genes in corneal endothelial samples derived from patients with FECD. We show that the majority of miRNA genes are hypermethylated at their promoter regions in FECD. More specifically, miR-199B is an extensively hypermethylated miRNA gene at its promoter region and its mature transcript miR-199b-5p was previously found to be almost completely silenced in FECD. Using a cell-based assay, we find that miR-199b-5p directly inhibits the expression of two epithelial mesenchymal transition (EMT)-inducing genes, Snai1 and ZEB1. Taken together, these findings suggest a novel regulatory mechanism of matrix protein production by corneal endothelial cells in which miR-199b-5p hypermethylation leads to its down-regulated expression and thereby the decreased expression of miR-199b-5p target genes, including Snai1 and ZEB1. Our results support miR-199b-5p as a potential therapeutic target to prevent or slow down the progression of FECD disease.\n\nAuthor summaryFuchs endothelial corneal dystrophy (FECD) due to corneal endothelial cell degeneration is one of the most common heritable causes of corneal visual loss and a leading indication for corneal transplantation. The progressive loss of corneal endothelial cells is accompanied by an abnormal deposition of extracellular matrix in the form of guttae. Here we discover that miRNA gene promoters are frequent targets of aberrant DNA methylation in FECD. In particular, we describe a novel epigenetic mechanism used by corneal endothelial cells to regulate extracellular matrix production. We find that miRNA-199b-5p functions as a negative regulator of Snai1 and ZEB1, two zinc finger transcription factors that have been shown to lead to increased production of extracellular matrix proteins. Furthermore, miR-199B was extensively hypermethylated in FECD and its mature transcript miR-199b-5p directly binds to the 3'-UTRs of Snai1 and ZEB1 genes. Ultimately, this may negatively modulate Snai1- and ZEB1-mediated production of extracellular matrix proteins. This work is the first to identify an important role of DNA methylation in the epigenetic regulation of miRNA-target genes in FECD and to describe a potential epigenetic biomarker for the treatment of FECD patients.

genetics↗