Citations

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Ex vivo CRISPR/Cas9‐mediated gene editing in hepatocytes using homology‐directed repair (HDR) is a potential alternative curative therapy to organ transplantation for metabolic liver disease. However, a major limitation of this approach in quiescent adult primary hepatocytes is that nonhomologous end‐joining is the predominant DNA repair pathway for double‐strand breaks (DSBs). This study explored the hypothesis that ex vivo hepatocyte culture could reprogram hepatocytes to favor HDR after CRISPR/Cas9‐mediated DNA DSBs. Quantitative PCR (qPCR), RNA sequencing, and flow cytometry demonstrated that within 24 hours, primary mouse hepatocytes in ex vivo monolayer culture decreased metabolic functions and increased expression of genes related to mitosis progression and HDR. Despite the down‐regulation of hepatocyte function genes, hepatocytes cultured for up to 72 hours could robustly engraft in vivo. To assess functionality long‐term, primary hepatocytes from a mouse model of hereditary tyrosinemia type 1 bearing a single‐point mutation were transduced ex vivo with two adeno‐associated viral vectors to deliver the Cas9 nuclease, target guide RNAs, and a 1.2‐kb homology template. Adeno‐associated viral Cas9 induced robust cutting at the target locus, and, after delivery of the repair template, precise correction of the point mutation occurred by HDR. Edited hepatocytes were transplanted into recipient fumarylacetoacetate hydrolase knockout mice, resulting in engraftment, robust proliferation, and prevention of liver failure. Weight gain and biochemical assessment revealed normalization of metabolic function. Conclusion: The results of this study demonstrate the potential therapeutic effect of ex vivo hepatocyte‐directed gene editing after reprogramming to cure metabolic disease in a preclinical model of hereditary tyrosinemia type 1.