IVT generation of guideRNAs for Cas9-enrichment Nanopore Sequencing.

Generating high-coverage sequencing coverage at select genomic loci has extensive applications in both research science and genetic medicine. Long-read sequencing technologies (e.g. nanopore sequencing) have expanded our ability to generate sequencing data in regions (e.g. repetitive elements) that are difficult to interrogate with short-read sequencing methods. In work presented here, we expand on our previous work using CRISPR/Cas9 for targeted nanopore sequencing by using in vitro transcribed guideRNAs, with 1100 guideRNAs in a single experiment. This approach decreases the cost per guideRNA, increases the number of guideRNAs that can be multiplexed in a single experiment, and provides a way to rapidly screen numerous guideRNAs for cutting efficiency. We apply this strategy in multiple patient-derived pancreatic cancer cell lines, demonstrating its ability to unveil structural variation in "deletion hotspots" around the tumor suppressor genes p16 (CDKN2A), and SMAD4.

Dysfunction of mitochondria and GABAergic interneurons in the anterior cingulate cortex of individuals with schizophrenia.

Here we re-analyze RNA-sequencing data from the anterior cingulate cortex (ACC) of SZ patients using recent methods to improve accuracy and sensitivity of results, such as the quality surrogate variable analysis (qSVA) method and the derfinder R package. We found that genes significantly down-regulated in SZ demonstrated an enrichment for parvalbumin-positive interneurons (FDR < 0.0001). Down-regulated genes were also enriched in oxidative phosphorylation functions (FDR < 0.05). We also addressed whether lifetime exposure to antipsychotics might influence gene expression, highlighting DUSP6, LBH, and NR1D1. Our results support the role of redox imbalance/mitochondrial dysfunction and implicate interneuron subtypes in SZ pathophysiology.

Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels.

OBJECTIVE: Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. Approach and Results: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) ß3, a downstream effector of Gαq. Activated PLCß3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCß3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. CONCLUSIONS: Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCß3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.

NCBI's Virus Discovery Codeathon: Building "FIVE" -The Federated Index of Viral Experiments API Index.

Viruses represent important test cases for data federation due to their genome size and the rapid increase in sequence data in publicly available databases. However, some consequences of previously decentralized (unfederated) data are lack of consensus or comparisons between feature annotations. Unifying or displaying alternative annotations should be a priority both for communities with robust entry representation and for nascent communities with burgeoning data sources. To this end, during this three-day continuation of the Virus Hunting Toolkit codeathon series (VHT-2), a new integrated and federated viral index was elaborated. This Federated Index of Viral Experiments (FIVE) integrates pre-existing and novel functional and taxonomy annotations and virus-host pairings. Variability in the context of viral genomic diversity is often overlooked in virus databases. As a proof-of-concept, FIVE was the first attempt to include viral genome variation for HIV, the most well-studied human pathogen, through viral genome diversity graphs. As per the publication of this manuscript, FIVE is the first implementation of a virus-specific federated index of such scope. FIVE is coded in BigQuery for optimal access of large quantities of data and is publicly accessible. Many projects of database or index federation fail to provide easier alternatives to access or query information. To this end, a Python API query system was developed to enhance the accessibility of FIVE.

Characterization of an unbalanced translocation causing 3q28qter duplication and 10q26.2qter deletion in a patient with global developmental delay and self-injury.

Chromosomal structural variation can cause severe neurodevelopmental and neuropsychiatric phenotypes. Here we present a nonverbal female adolescent with severe stereotypic movement disorder with severe problem behavior (e.g., self-injurious behavior, aggression, and disruptive and destructive behaviors), autism spectrum disorder, severe intellectual disability, attention deficit hyperactivity disorder, and global developmental delay. Previous cytogenetic analysis revealed balanced translocations present in the patient's apparently normal mother. We hypothesized the presence of unbalanced translocations in the patient due to maternal history of spontaneous abortions. Whole-genome sequencing and whole-genome optical mapping, complementary next-generation genomic technologies capable of the accurate and robust detection of structural variants, identified t(3;10), t(10;14), and t(3;14) three-way balanced translocations in the mother and der(10)t(3;14;10) and der(14)t(3;14;10) translocations in the patient. Instead of a t(3;10), she inherited a normal maternal copy of Chromosome 3, resulting in an unbalanced state of a 3q28qter duplication and 10q26.2qter deletion. Copy-imbalanced genes in one or both of these regions, such as DLG1, DOCK1, and EBF3, may contribute to the patient's phenotype that spans neurodevelopmental, musculoskeletal, and psychiatric domains, with the possible contribution of a maternally inherited 15q13.2q13.3 deletion.

Author Correction: Template plasmid integration in germline genome-edited cattle.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increased Protein Insolubility in Brains From a Subset of Patients With Schizophrenia.

OBJECTIVE: The mechanisms leading to schizophrenia are likely to be diverse. However, there may be common pathophysiological pathways for subtypes of the disease. The authors tested the hypothesis that increased protein insolubility and ubiquitination underlie the pathophysiology for a subtype of schizophrenia. METHODS: Prefrontal cortex and superior temporal gyrus from postmortem brains of individuals with and without schizophrenia were subjected to cold sarkosyl fractionation, separating proteins into soluble and insoluble fractions. Protein insolubility and ubiquitin levels were quantified for each insoluble fraction, with normalization to total homogenate protein. Mass spectrometry analysis was then performed to identify the protein contents of the insoluble fractions. The potential biological relevance of the detected proteins was assessed using Gene Ontology enrichment analysis and Ingenuity Pathway Analysis. RESULTS: A subset of the schizophrenia brains showed an increase in protein insolubility and ubiquitination in the insoluble fraction. Mass spectrometry of the insoluble fraction revealed that brains with increased insolubility and ubiquitination exhibited a similar peptide expression by principal component analysis. The proteins that were significantly altered in the insoluble fraction were enriched for pathways relating to axon target recognition as well as nervous system development and function. CONCLUSIONS: This study suggests a pathological process related to protein insolubility for a subset of patients with schizophrenia. Determining the molecular mechanism of this subtype of schizophrenia could lead to a better understanding of the pathways underlying the clinical phenotype in some patients with major mental illness as well as to improved nosology and identification of novel therapeutic targets.

Validation Strategy for Ultrasensitive Mutation Detection.

BACKGROUND: Ultrasensitive detection of low-abundance DNA point mutations is a challenging molecular biology problem, because nearly identical mutant and wild-type molecules exhibit crosstalk. Reliable ultrasensitive point mutation detection will facilitate early detection of cancer and therapeutic monitoring of cancer patients. OBJECTIVE: The objective of this study was to develop a method to correct errors in low-level cell line mixes. MATERIALS AND METHODS: We tested sample mixes with digital-droplet PCR (ddPCR) and next-generation sequencing. RESULTS: We introduced two corrections: baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation; and haplotype counting was used to correct errors in cell counting and pipetting. We found ddPCR to have better correlation for detecting low-level mutations without applying any correction (R2 = 0.80) and be more linear after introducing both corrections (R2 = 0.99). CONCLUSIONS: The VAF correction was found to be more significant than haplotype correction. It is imperative that various technologies be evaluated against each other and laboratories be provided with defined quality control samples for proficiency testing.

qSVA framework for RNA quality correction in differential expression analysis.

RNA sequencing (RNA-seq) is a powerful approach for measuring gene expression levels in cells and tissues, but it relies on high-quality RNA. We demonstrate here that statistical adjustment using existing quality measures largely fails to remove the effects of RNA degradation when RNA quality associates with the outcome of interest. Using RNA-seq data from molecular degradation experiments of human primary tissues, we introduce a method-quality surrogate variable analysis (qSVA)-as a framework for estimating and removing the confounding effect of RNA quality in differential expression analysis. We show that this approach results in greatly improved replication rates (>3×) across two large independent postmortem human brain studies of schizophrenia and also removes potential RNA quality biases in earlier published work that compared expression levels of different brain regions and other diagnostic groups. Our approach can therefore improve the interpretation of differential expression analysis of transcriptomic data from human tissue.

Analysis of differential gene expression mediated by clozapine in human postmortem brains.

Clozapine is the only medication indicated for treating refractory schizophrenia, due to its superior efficacy among all antipsychotic agents, but its mechanism of action is poorly understood. To date, no studies of human postmortem brain have characterized the gene expression response to clozapine. Therefore, we addressed this question by analyzing expression data extracted from published microarray studies involving brains of patients on antipsychotic therapy. We first performed a systematic review and identified four microarray studies of postmortem brains from antipsychotic-treated patients, then extracted the expression data. We then performed generalized linear model analysis on each study separately, and identified the genes differentially expressed in response to clozapine compared to other atypical antipsychotic medications, as well as their associated canonical pathways. We also found a number of genes common to all four studies that we analyzed: GCLM, ZNF652, and GYPC. In addition, pathway analysis highlighted the following processes in all four studies: clathrin-mediated endocytosis, SAPK/JNK signaling, 3-phosphoinositide synthesis, and paxillin signaling. Our analysis yielded the first comprehensive compendium of genes and pathways differentially expressed upon clozapine treatment in the human brain, which may provide insight into the mechanism and unique efficacy of clozapine, as well as the pathophysiology of schizophrenia.

Nanopore sequencing detects structural variants in cancer.

Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300 bp) of 2nd generation sequencing. Not only do the reads (or paired-end reads) need to straddle a breakpoint, but repetitive elements often lead to ambiguities in the alignment of short reads. We propose to use the long-reads (up to 20 kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Though nanopore sequencing currently has a relatively high mismatch rate that precludes base substitution and small frameshift mutation detection, its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency. We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.

Whole Genome Sequencing Defines the Genetic Heterogeneity of Familial Pancreatic Cancer.

UNLABELLED: Pancreatic cancer is projected to become the second leading cause of cancer-related death in the United States by 2020. A familial aggregation of pancreatic cancer has been established, but the cause of this aggregation in most families is unknown. To determine the genetic basis of susceptibility in these families, we sequenced the germline genomes of 638 patients with familial pancreatic cancer and the tumor exomes of 39 familial pancreatic adenocarcinomas. Our analyses support the role of previously identified familial pancreatic cancer susceptibility genes such as BRCA2, CDKN2A, and ATM, and identify novel candidate genes harboring rare, deleterious germline variants for further characterization. We also show how somatic point mutations that occur during hematopoiesis can affect the interpretation of genome-wide studies of hereditary traits. Our observations have important implications for the etiology of pancreatic cancer and for the identification of susceptibility genes in other common cancer types. SIGNIFICANCE: The genetic basis of disease susceptibility in the majority of patients with familial pancreatic cancer is unknown. We whole genome sequenced 638 patients with familial pancreatic cancer and demonstrate that the genetic underpinning of inherited pancreatic cancer is highly heterogeneous. This has significant implications for the management of patients with familial pancreatic cancer.

Transflip mutations produce deletions in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is driven by the inactivation of the tumor suppressor genes (TSGs), CDKN2A (P16) and SMAD4 (DPC4), commonly by homozygous deletions (HDs). Using a combination of high density single-nucleotide polymorphism (SNP) microarray and whole genome sequencing (WGS), we fine-mapped novel breakpoints surrounding deletions of CDKN2A and SMAD4 and characterized them by their underlying structural variants (SVs). Only one third of CDKN2A and SMAD4 deletions (6 of 18) were simple interstitial deletions, rather, the majority of deletions were caused by complex rearrangements, specifically, a translocation on one side of the TSG in combination with an inversion on the other side. We designate these as "TransFlip" mutations. Characteristics of TransFlip mutations are: (1) a propensity to target the TSGs CDKN2A and SMAD4 (P < 0.005), (2) not present in the germline of the examined samples, (3) non-recurrent breakpoints, (4) relatively small (47 bp to 3.4 kb) inversions, (5) inversions can be either telomeric or centromeric to the TSG, and (6) non-reciprocal, and non-recurrent translocations. TransFlip mutations are novel complex genomic rearrangements with unique breakpoint signatures in pancreatic cancer. We hypothesize that they are a common but poorly understood mechanism of TSG inactivation in human cancer. © 2015 Wiley Periodicals, Inc.

Familial and sporadic pancreatic cancer share the same molecular pathogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is nearly uniformly lethal, with a median overall survival in 2014 of only 6 months. The genetic progression of sporadic PDAC (SPC) is well established, with common somatic alterations in KRAS, p16/CDKN2A, TP53, and SMAD4/DPC4. Up to 10 % of all PDAC cases occur in families with two or more affected first-degree relatives (familial pancreatic cancer, FPC), but these cases do not appear to present at an obviously earlier age of onset. This is unusual because most familial cancer syndrome patients present at a substantially younger age than that of corresponding sporadic cases. Here we collated the reported age of onset for FPC and SPC from the literature. We then used an integrated approach including whole exomic sequencing, whole genome sequencing, RNA sequencing, and high density SNP microarrays to study a cohort of FPC cell lines and corresponding germline samples. We show that the four major SPC driver genes are also consistently altered in FPC and that each of the four detection strategies was able to detect the mutations in these genes, with one exception. We conclude that FPC undergoes a similar somatic molecular pathogenesis as SPC, and that the same gene targets can be used for early detection and minimal residual disease testing in FPC patients.

KRAS and guanine nucleotide-binding protein mutations in pancreatic juice collected from the duodenum of patients at high risk for neoplasia undergoing endoscopic ultrasound.

BACKGROUND & AIMS: Pancreatic imaging can identify neoplastic cysts but not microscopic neoplasms. Mutation analysis of pancreatic fluid after secretin stimulation might identify microscopic neoplasias in the pancreatic duct system. We determined the prevalence of mutations in KRAS and guanine nucleotide-binding protein α-stimulating genes in pancreatic juice from subjects undergoing endoscopic ultrasound for suspected pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, or pancreatic adenocarcinoma. METHODS: Secretin-stimulated juice samples were collected from the duodenum of 272 subjects enrolled in Cancer of the Pancreas Screening studies; 194 subjects were screened because of a family history of, or genetic predisposition to, pancreatic cancer, and 78 subjects were evaluated for pancreatic cancer (n = 30) or other disorders (controls: pancreatic cysts, pancreatitis, or normal pancreata, n = 48). Mutations were detected by digital high-resolution melt-curve analysis and pyrosequencing. The number of replicates containing a mutation determined the mutation score. RESULTS: KRAS mutations were detected in pancreatic juice from larger percentages of subjects with pancreatic cancer (73%) or undergoing cancer screening (50%) than controls (19%) (P = .0005). A greater proportion of patients with pancreatic cancer had at least 1 KRAS mutation detected 3 or more times (47%) than screened subjects (21%) or controls (6%, P = .002). Among screened subjects, mutations in KRAS (but not guanine nucleotide-binding protein α-stimulating) were found in similar percentages of patients with or without pancreatic cysts. However, a greater proportion of patients older than age 50 years had KRAS mutations (54.6%) than younger patients (36.3%) (P = .032); the older subjects also had more mutations in KRAS (P = .02). CONCLUSIONS: Mutations in KRAS are detected in pancreatic juice from the duodenum of 73% of patients with pancreatic cancer, and 50% of asymptomatic individuals with a high risk for pancreatic cancer. However, KRAS mutations were detected in pancreatic juice from 19% of controls. Mutations detected in individuals without pancreatic abnormalities, based on imaging analyses, likely arise from small pancreatic intraepithelial neoplasia lesions. ClinicalTrials.gov no: NCT00438906 and NCT00714701.

Clinicopathological characteristics and molecular analyses of multifocal intraductal papillary mucinous neoplasms of the pancreas.

OBJECTIVE: To examine the clinicopathologic features and clonal relationship of multifocal intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. BACKGROUND: Intraductal papillary mucinous neoplasms are increasingly diagnosed cystic precursor lesions of pancreatic cancer. Intraductal papillary mucinous neoplasms can be multifocal and a potential cause of recurrence after partial pancreatectomy. METHODS: Thirty four patients with histologically documented multifocal IPMNs were collected and their clinicopathologic features catalogued. In addition, thirty multifocal IPMNs arising in 13 patients from 3 hospitals were subjected to laser microdissection followed by KRAS pyrosequencing and loss of heterozygosity (LOH) analysis on chromosomes 6q and 17p. Finally, we sought to assess the clonal relationships among multifocal IPMNs. RESULTS: We identified 34 patients with histologically documented multifocal IPMNs. Synchronous IPMNs were present in 29 patients (85%), whereas 5 (15%) developed clinically significant metachronous IPMNs. Six patients (18%) had a history of familial pancreatic cancer. A majority of multifocal IPMNs (86% synchronous, 100% metachronous) were composed of branch duct lesions, and typically demonstrated a gastric-foveolar subtype epithelium with low or intermediate grades of dysplasia. Three synchronous IPMNs (10%) had an associated invasive cancer. Molecular analysis of multiple IPMNs from 13 patients demonstrated nonoverlapping KRAS gene mutations in 8 patients (62%) and discordant LOH profiles in 7 patients (54%); independent genetic alterations were established in 9 of the 13 patients (69%). CONCLUSIONS: The majority of multifocal IPMNs arise independently and exhibit a gastric-foveolar subtype, with low to intermediate dysplasia. These findings underscore the importance of life-long follow-up after resection for an IPMN.