Genetic correlations and an increased incidence of psychiatric disorders in inflammatory-bowel disease have been reported, but shared molecular mechanisms are unknown. We performed cross-tissue and multiple-gene conditioned transcriptome-wide association studies for 23 tissues of the gut-brain-axis using genome-wide association studies data sets (total 180,592 patients) for Crohn’s disease, ulcerative colitis, primary sclerosing cholangitis, schizophrenia, bipolar disorder, major depressive disorder and attention-deficit/hyperactivity disorder. We identified NR5A2, SATB2, and PPP3CA (encoding a target for calcineurin inhibitors in refractory ulcerative colitis) as shared susceptibility genes with transcriptome-wide significance both for Crohn’s disease, ulcerative colitis and schizophrenia, largely explaining fine-mapped association signals at nearby genome-wide association study susceptibility loci. Analysis of bulk and single-cell RNA-sequencing data showed that PPP3CA expression was strongest in neurons and in enteroendocrine and Paneth-like cells of the ileum, colon, and rectum, indicating a possible link to the gut-brain-axis. PPP3CA together with three further suggestive loci can be linked to calcineurin-related signaling pathways such as NFAT activation or Wnt.
In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called ‘ERROR-FFPE-DNA’ that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.
Background and Aims Inflammatory bowel disease (IBD) is a chronic relapsing disorder of the gastrointestinal tract, which generally manifests as Crohn’s disease (CD) or ulcerative colitis (UC). These subtypes are heterogeneous in terms of disease location and histological features, while sharing common clinical presentation, genetic associations and thus, common immune regulatory pathways. Methods Using miRNA and mRNA coupled transcriptome profiling and systems biology approaches, we report a comprehensive analysis of blood transcriptomes from treatment-naïve (n=110) and treatment-exposed (n=177) IBD patients as well as symptomatic- (n=65) and healthy controls (n=95). Results Broadly, the peripheral blood transcriptomes of CD and UC patients were similar. However, there was an extensive gene deregulation in the blood of IBD patients, while only a slight deregulation in symptomatic controls, when compared with healthy controls. The deregulated mRNAs and miRNAs are mainly involved in the innate immunity and are especially enriched in neutrophil activation-related pathways. Oxidative phosphorylation and neutrophil activation-related modules were found to be differentially co-expressed among treatment-naïve IBD as compared to healthy controls. In the deregulated neutrophil activation-related co-expression module, the IL1B was identified as the central gene. The co-expression levels among IL1B and chemosensing receptor (CXCR1/2 and FPR1/2) genes were reduced in the blood of IBD patients when compared with healthy controls. Conclusions Immune dysregulation seen in peripheral blood transcriptomes of treatment-naïve IBD patients is mainly driven by neutrophil activation.
Hybridisation-based targeted enrichment is a widely used and well-established technique in high-throughput second-generation short-read sequencing. Despite the high potential to genetically resolve highly repetitive and variable genomic sequences by, for example PacBio third-generation sequencing, targeted enrichment for long fragments has not yet established the same high-throughput due to currently existing complex workflows and technological dependencies. We here describe a scalable targeted enrichment protocol for fragment sizes of >7 kb. For demonstration purposes we developed a custom blood group panel of challenging loci. Test results achieved > 65% on-target rate, good coverage (142.7×) and sufficient coverage evenness for both non-paralogous and paralogous targets, and sufficient non-duplicate read counts (83.5%) per sample for a highly multiplexed enrichment pool of 16 samples. We genotyped the blood groups of nine patients employing highly accurate phased assemblies at an allelic resolution that match reference blood group allele calls determined by SNP array and NGS genotyping. Seven Genome-in-a-Bottle reference samples achieved high recall (96%) and precision (99%) rates. Mendelian error rates were 0.04% and 0.13% for the included Ashkenazim and Han Chinese trios, respectively. In summary, we provide a protocol and first example for accurate targeted long-read sequencing that can be used in a high-throughput fashion.
Liquid biopsies are a minimally invasive method to diagnose and longitudinally monitor tumor mutations in patients when tissue biopsies are difficult (e.g., in lung cancer). The percentage of cell-free tumor DNA in blood plasma ranges from more than 65% to 0.1% or lower. To reliably diagnose tumor mutations at 0.1%, there are two options: unrealistically large volumes of patient blood or library preparation and sequencing depth optimized to low-input DNA. Here, we assess two library preparation methods and analysis workflows to determine feasibility and reliability based on standards with known allelic frequency (0 and 0.13% in PIK3CA). However, the implementation for patients is still costly and requires elaborate setups.
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