SCHOOL: Software for Clinical Health in Oncology for Omics Laboratories.

Bioinformatics analysis is a key element in the development of in-house next-generation sequencing assays for tumor genetic profiling that can include both tumor DNA and RNA with comparisons to matched-normal DNA in select cases. Bioinformatics analysis encompasses a computationally heavy component that requires a high-performance computing component and an assay-dependent quality assessment, aggregation, and data cleaning component. Although there are free, open-source solutions and fee-for-use commercial services for the computationally heavy component, these solutions and services can lack the options commonly utilized in increasingly complex genomic assays. Additionally, the cost to purchase commercial solutions or implement and maintain open-source solutions can be out of reach for many small clinical laboratories. Here, we present Software for Clinical Health in Oncology for Omics Laboratories (SCHOOL), a collection of genomics analysis workflows that (i) can be easily installed on any platform; (ii) run on the cloud with a user-friendly interface; and (iii) include the detection of single nucleotide variants, insertions/deletions, copy number variants (CNVs), and translocations from RNA and DNA sequencing. These workflows contain elements for customization based on target panel and assay design, including somatic mutational analysis with a matched-normal, microsatellite stability analysis, and CNV analysis with a single nucleotide polymorphism backbone. All of the features of SCHOOL have been designed to run on any computer system, where software dependencies have been containerized. SCHOOL has been built into apps with workflows that can be run on a cloud platform such as DNANexus using their point-and-click graphical interface, which could be automated for high-throughput laboratories.

ANSWER: Annotation Software for Electronic Reporting.

PURPOSE: To better use genetic testing, which is used by clinicians to explain the molecular mechanism of disease and to suggest clinical actionability and new treatment options, clinical next-generation sequencing (NGS) laboratories must send the results into reports in PDF and discrete data element format (HL7). Although most clinical diagnostic tests have set molecular markers tested and have a set range of values or a binary result (positive or negative), the NGS genetic test could examine hundreds or thousands of genes with no predefined list of variants. Although there are some commercial and open-source tools for clinically reporting genomics results for oncology testing, they often lack necessary features. METHODS: Using several available software tools for data storage including MySQL and MongoDB, database querying with Python, and a web-based user application using JAVA and JAVA script, we have developed a tool to store and query complex genomics and demographics data, which can be manually curated and reported by the user. RESULTS: We have developed a tool, Annotation SoftWare for Electronic Reporting (ANSWER), that can allow molecular pathologists to (1) filter variants to find those meeting quality control metrics in the genes that are clinically actionable by diagnosis; (2) visualize variants using data generated in the bioinformatics analysis; (3) create annotations that can be reused in future reports with association specific to the gene, variant, or diagnosis; (4) select variants and annotations that should be reported to match the details of the case; and (5) generate a report that includes demographics, reported variants, clinical actionability annotation, and references that can be exported into PDF or HL7 format, which can be electronically sent to an electronic health record. CONCLUSION: ANSWER is a tool that can be installed locally and is designed to meet the clinical reporting needs of a clinical oncology NGS laboratory for reporting.

IRF1 governs the differential interferon-stimulated gene responses in human monocytes and macrophages by regulating chromatin accessibility.

Myeloid lineage cells use TLRs to recognize and respond to diverse microbial ligands. Although unique transcription factors dictate the outcome of specific TLR signaling, whether lineage-specific differences exist to further modulate the quality of TLR-induced inflammation remains unclear. Comprehensive analysis of global gene transcription in human monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells stimulated with various TLR ligands identifies multiple lineage-specific, TLR-responsive gene programs. Monocytes are hyperresponsive to TLR7/8 stimulation that correlates with the higher expression of the receptors. While macrophages and monocytes express similar levels of TLR4, macrophages, but not monocytes, upregulate interferon-stimulated genes (ISGs) in response to TLR4 stimulation. We find that TLR4 signaling in macrophages uniquely engages transcription factor IRF1, which facilitates the opening of ISG loci for transcription. This study provides a critical mechanistic basis for lineage-specific TLR responses and uncovers IRF1 as a master regulator for the ISG transcriptional program in human macrophages.

Deep sequencing reveals a DAP1 regulatory haplotype that potentiates autoimmunity in systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease characterized by the development of anti-nuclear antibodies. Susceptibility to SLE is multifactorial, with a combination of genetic and environmental risk factors contributing to disease development. Like other polygenic diseases, a significant proportion of estimated SLE heritability is not accounted for by common disease alleles analyzed by SNP array-based GWASs. Death-associated protein 1 (DAP1) was implicated as a candidate gene in a previous familial linkage study of SLE and rheumatoid arthritis, but the association has not been explored further. RESULTS: We perform deep sequencing across the DAP1 genomic segment in 2032 SLE patients, and healthy controls, and discover a low-frequency functional haplotype strongly associated with SLE risk in multiple ethnicities. We find multiple cis-eQTLs embedded in a risk haplotype that progressively downregulates DAP1 transcription in immune cells. Decreased DAP1 transcription results in reduced DAP1 protein in peripheral blood mononuclear cells, monocytes, and lymphoblastoid cell lines, leading to enhanced autophagic flux in immune cells expressing the DAP1 risk haplotype. Patients with DAP1 risk allele exhibit significantly higher autoantibody titers and altered expression of the immune system, autophagy, and apoptosis pathway transcripts, indicating that the DAP1 risk allele mediates enhanced autophagy, leading to the survival of autoreactive lymphocytes and increased autoantibody. CONCLUSIONS: We demonstrate how targeted sequencing captures low-frequency functional risk alleles that are missed by SNP array-based studies. SLE patients with the DAP1 genotype have distinct autoantibody and transcription profiles, supporting the dissection of SLE heterogeneity by genetic analysis.

Co-circulation dynamics and persistence of newly introduced clades of 2012 outbreak associated West Nile Virus in Texas, 2012-2015.

The second largest outbreak of West Nile encephalitis and West Nile fever ever recorded occurred in the United States (U.S) in the summer of 2012. The outbreak was related to the widespread circulation of closely related clades, or groups, of West Nile virus (WNV) into multiple states where they were not previously found. Whether the invading 2012 strains were able to circulate and overwinter in states with their own endemic population of WNV is unknown and the effect of viral genetics on adaptation and persistence in a new ecological niche is unclear. In this study, we sequenced 70 mosquito isolates from multiple counties throughout Texas in 2012-2015. We identified isolates representative of previously described 2012 WNV groups (Groups 8-10) and discovered a novel group which we called Group 11. Although we identified isolates representative of WNV endemic (2/70) to Texas, most isolates (68/70) were related to the invading 2012 strains, and of these Group 10 (45/68) was predominant. We also observed differences among the 2012 WNV groups correlating to their genotype. Group 10 WNV in Texas, which carry two putative positively selected variants, had limited introductions into Texas, wide circulation, and strong evidence of continued persistence perhaps indicative of overwintering. In contrast, Groups 8 and 11, without positively selected variants, had multiple introductions into Texas, limited circulation, and limited persistence. Lastly, we identified a potential transmission source in New York for incoming Group 8 WNV into Texas. Altogether our study suggests that mutations in the WNV genome may influence the range and dynamics of WNV circulation, and the ability of different strains to persist in new ecological niches.

Regulatory polymorphisms modulate the expression of HLA class II molecules and promote autoimmunity.

Targeted sequencing of sixteen SLE risk loci among 1349 Caucasian cases and controls produced a comprehensive dataset of the variations causing susceptibility to systemic lupus erythematosus (SLE). Two independent disease association signals in the HLA-D region identified two regulatory regions containing 3562 polymorphisms that modified thirty-seven transcription factor binding sites. These extensive functional variations are a new and potent facet of HLA polymorphism. Variations modifying the consensus binding motifs of IRF4 and CTCF in the XL9 regulatory complex modified the transcription of HLA-DRB1, HLA-DQA1 and HLA-DQB1 in a chromosome-specific manner, resulting in a 2.5-fold increase in the surface expression of HLA-DR and DQ molecules on dendritic cells with SLE risk genotypes, which increases to over 4-fold after stimulation. Similar analyses of fifteen other SLE risk loci identified 1206 functional variants tightly linked with disease-associated SNPs and demonstrated that common disease alleles contain multiple causal variants modulating multiple immune system genes.

DGKE variants cause a glomerular microangiopathy that mimics membranoproliferative GN.

Renal microangiopathies and membranoproliferative GN (MPGN) can manifest similar clinical presentations and histology, suggesting the possibility of a common underlying mechanism in some cases. Here, we performed homozygosity mapping and whole exome sequencing in a Turkish consanguineous family and identified DGKE gene variants as the cause of a membranoproliferative-like glomerular microangiopathy. Furthermore, we identified two additional DGKE variants in a cohort of 142 unrelated patients diagnosed with membranoproliferative GN. This gene encodes the diacylglycerol kinase DGKε, which is an intracellular lipid kinase that phosphorylates diacylglycerol to phosphatidic acid. Immunofluorescence confocal microscopy demonstrated that mouse and rat Dgkε colocalizes with the podocyte marker WT1 but not with the endothelial marker CD31. Patch-clamp experiments in human embryonic kidney (HEK293) cells showed that DGKε variants affect the intracellular concentration of diacylglycerol. Taken together, these results not only identify a genetic cause of a glomerular microangiopathy but also suggest that the phosphatidylinositol cycle, which requires DGKE, is critical to the normal function of podocytes.

Association of two independent functional risk haplotypes in TNIP1 with systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and altered type I interferon expression. Genetic surveys and genome-wide association studies have identified >30 SLE susceptibility genes. One of these genes, TNIP1, encodes the ABIN1 protein. ABIN1 functions in the immune system by restricting NF-κB signaling. The present study was undertaken to investigate the genetic factors that influence association with SLE in genes that regulate the NF-κB pathway. METHODS: We analyzed a dense set of genetic markers spanning TNIP1 and TAX1BP1, as well as the TNIP1 homolog TNIP2, in case-control populations of diverse ethnic origins. TNIP1, TNIP2, and TAX1BP1 were fine-mapped in a total of 8,372 SLE cases and 7,492 healthy controls from European-ancestry, African American, Hispanic, East Asian, and African American Gullah populations. Levels of TNIP1 messenger RNA (mRNA) and ABIN1 protein in Epstein-Barr virus-transformed human B cell lines were analyzed by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: We found significant associations between SLE and genetic variants within TNIP1, but not in TNIP2 or TAX1BP1. After resequencing and imputation, we identified 2 independent risk haplotypes within TNIP1 in individuals of European ancestry that were also present in African American and Hispanic populations. Levels of TNIP1 mRNA and ABIN1 protein were reduced among subjects with these haplotypes, suggesting that they harbor hypomorphic functional variants that influence susceptibility to SLE by restricting ABIN1 expression. CONCLUSION: Our results confirm the association signals between SLE and TNIP1 variants in multiple populations and provide new insight into the mechanism by which TNIP1 variants may contribute to SLE pathogenesis.

Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study.

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p(meta-Euro) = 2.08 × 10(-10)), transmembrane protein 39A (TMEM39A; rs1132200; p(meta-all) = 8.62 × 10(-9)), and 17q21 (rs1453560; p(meta-all) = 3.48 × 10(-10)) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10(-8) < p(meta-Euro) < 9.99 × 10(-5)) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.