Protective effect of PDE4B subtype-specific inhibition in an App knock-in mouse model for Alzheimer's disease.

Meta-analysis of genome-wide association study data has implicated PDE4B in the pathogenesis of Alzheimer's disease (AD), the leading cause of senile dementia. PDE4B encodes one of four subtypes of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase-4 (PDE4A-D). To interrogate the involvement of PDE4B in the manifestation of AD-related phenotypes, the effects of a hypomorphic mutation (Pde4bY358C) that decreases PDE4B's cAMP hydrolytic activity were evaluated in the AppNL-G-F knock-in mouse model of AD using the Barnes maze test of spatial memory, 14C-2-deoxyglucose autoradiography, thioflavin-S staining of ß-amyloid (Aß) plaques, and inflammatory marker assay and transcriptomic analysis (RNA sequencing) of cerebral cortical tissue. At 12 months of age, AppNL-G-F mice exhibited spatial memory and brain metabolism deficits, which were prevented by the hypomorphic PDE4B in AppNL-G-F/Pde4bY358C mice, without a decrease in Aß plaque burden. RNA sequencing revealed that, among the 531 transcripts differentially expressed in AppNL-G-F versus wild-type mice, only 13 transcripts from four genes - Ide, Btaf1, Padi2, and C1qb - were differentially expressed in AppNL-G-F/Pde4bY358C versus AppNL-G-F mice, identifying their potential involvement in the protective effect of hypomorphic PDE4B. Our data demonstrate that spatial memory and cerebral glucose metabolism deficits exhibited by 12-month-old AppNL-G-F mice are prevented by targeted inhibition of PDE4B. To our knowledge, this is the first demonstration of a protective effect of PDE4B subtype-specific inhibition in a preclinical model of AD. It thus identifies PDE4B as a key regulator of disease manifestation in the AppNL-G-F model and a promising therapeutic target for AD.

A recurrent de novo MAX p.Arg60Gln variant causes a syndromic overgrowth disorder through differential expression of c-Myc target genes.

Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.

Transcriptome profiles of stem-like cells from primary breast cancers allow identification of ITGA7 as a predictive marker of chemotherapy response.

BACKGROUND: Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. METHODS: Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. RESULTS: Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. CONCLUSIONS: This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

Targeting human plasmacytoid dendritic cells through BDCA2 prevents skin inflammation and fibrosis in a novel xenotransplant mouse model of scleroderma.

OBJECTIVES: Plasmacytoid dendritic cells (pDC) have been implicated in the pathogenesis of autoimmune diseases, such as scleroderma (SSc). However, this has been derived from indirect evidence using ex vivo human samples or mouse pDC in vivo. We have developed human-specific pDC models to directly identify their role in inflammation and fibrosis, as well as attenuation of pDC function with BDCA2-targeting to determine its therapeutic application. METHODS: RNAseq of human pDC with TLR9 agonist ODN2216 and humanised monoclonal BDCA2 antibody, CBS004. Organotypic skin rafts consisting of fibroblasts and keratinocytes were stimulated with supernatant from TLR9-stimulated pDC and with CBS004. Human pDC were xenotransplanted into Nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) with CBS004 or human IgG control. Skin punch biopsies were used to assess gene and protein expression. RESULTS: RNAseq shows TLR9-induced activation of human pDC goes beyond type I interferon (IFN) secretion, which is functionally inactivated by BDCA2-targeting. Consistent with these findings, we show that BDCA2-targeting of pDC can completely suppress in vitro skin IFN-induced response. Most importantly, xenotransplantation of human pDC significantly increased in vivo skin IFN-induced response to TLR agonist and strongly enhanced fibrotic and immune response to bleomycin compared with controls. In these contexts, BDCA2-targeting suppressed human pDC-specific pathological responses. CONCLUSIONS: Our data indicate that human pDC play a key role in inflammation and immune-driven skin fibrosis, which can be effectively blocked by BDCA2-targeting, providing direct evidence supporting the development of attenuation of pDC function as a therapeutic application for SSc.

Assessing the utility of long-read nanopore sequencing for rapid and efficient characterization of mobile element insertions.

Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. "Third-generation" long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Long-read nanopore sequencing was then performed on locus-specific amplicons. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing.

Effects of quercetin and metabolites on uric acid biosynthesis and consequences for gene expression in the endothelium.

BACKGROUND: Uric acid, a metabolic product of purine degradation in humans, is a risk factor for developing gout and type 2 diabetes, and supplementation with quercetin lowers plasma uric acid in mildly hyperuricemic men. Here we examined the mechanism of inhibition of enzymes involved in uric acid metabolism by quercetin, conjugates and microbial catabolites, and measured the effect of lowered circulating uric acid on endothelial cell gene expression. METHODS: Inhibition of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and xanthine oxidoreductase (XOR) activity by quercetin and metabolites was determined by HPLC. Human umbilical vein endothelial cells (HUVECs) were cultured under conditions mimicking blood flow, treated with uric acid (0, 300 or 500 µmol/L), and changes in gene expression measured using transcriptomics and quantitative droplet digital PCR. RESULTS: In human plasma, no inhibition of PNP activity was observed, and only quercetin weakly inhibited ADA. XOR was not present at sufficient amount in human plasma to use for testing, but quercetin, quercetin-3'-sulfate and the gut microbial metabolite 3',4'-dihydroxyphenylacetic acid inhibited bovine milk XOR. Several changes were observed in gene expression in HUVECs under flow compared to static conditions, but after uric acid treatment, only very few changes were detected. CONCLUSIONS: We propose that the main mechanism by which quercetin, as quercetin-3'-sulfate, lowers uric acid in vivo is through inhibition of XOR, and not ADA nor PNP. The pertinent shift in uric acid concentration was not sufficient to produce significant changes in endothelial gene expression in a cell model.

Functionally impaired plasmacytoid dendritic cells and non-haematopoietic sources of type I interferon characterize human autoimmunity.

Autoimmune connective tissue diseases arise in a stepwise fashion from asymptomatic preclinical autoimmunity. Type I interferons have a crucial role in the progression to established autoimmune diseases. The cellular source and regulation in disease initiation of these cytokines is not clear, but plasmacytoid dendritic cells have been thought to contribute to excessive type I interferon production. Here, we show that in preclinical autoimmunity and established systemic lupus erythematosus, plasmacytoid dendritic cells are not effector cells, have lost capacity for Toll-like-receptor-mediated cytokine production and do not induce T cell activation, independent of disease activity and the blood interferon signature. In addition, plasmacytoid dendritic cells have a transcriptional signature indicative of cellular stress and senescence accompanied by increased telomere erosion. In preclinical autoimmunity, we show a marked enrichment of an interferon signature in the skin without infiltrating immune cells, but with interferon-κ production by keratinocytes. In conclusion, non-hematopoietic cellular sources, rather than plasmacytoid dendritic cells, are responsible for interferon production prior to clinical autoimmunity.

Translating Biomarkers of Cholangiocarcinoma for Theranosis: A Systematic Review.

Cholangiocarcinoma (CCA) is a rare disease with poor outcomes and limited research efforts into novel treatment options. A systematic review of CCA biomarkers was undertaken to identify promising biomarkers that may be used for theranosis (therapy and diagnosis). MEDLINE/EMBASE databases (1996-2019) were systematically searched using two strategies to identify biomarker studies of CCA. The PANTHER Go-Slim classification system and STRING network version 11.0 were used to interrogate the identified biomarkers. The TArget Selection Criteria for Theranosis (TASC-T) score was used to rank identified proteins as potential targetable biomarkers for theranosis. The following proteins scored the highest, CA9, CLDN18, TNC, MMP9, and EGFR, and they were evaluated in detail. None of these biomarkers had high sensitivity or specificity for CCA but have potential for theranosis. This review is unique in that it describes the process of selecting suitable markers for theranosis, which is also applicable to other diseases. This has highlighted existing validated markers of CCA that can be used for active tumor targeting for the future development of targeted theranostic delivery systems. It also emphasizes the relevance of bioinformatics in aiding the search for validated biomarkers that could be repurposed for theranosis.

The Ability of Quercetin and Ferulic Acid to Lower Stored Fat is Dependent on the Metabolic Background of Human Adipocytes.

SCOPE: Dietary flavonoids and phenolic acids can modulate lipid metabolism, but effects on mature human adipocytes are not well characterized. MATERIALS AND METHODS: Human adipocytes are differentiated, and contain accumulated lipids, mimicking white adipocytes. They are then cultured either under conditions of actively synthesizing and accumulating additional lipids through lipogenesis ("ongoing lipogenic state") or under conditions of maintaining but not increasing stored lipids ("lipid storage state"). Total lipid, lipidomic and transcriptomics analyses are employed to assess changes after treatment with quercetin and/or ferulic acid. RESULTS: In the "lipid storage state," a longer-term treatment (3 doses over 72 h) with low concentrations of quercetin and ferulic acid together significantly lowered stored lipid content, modified lipid composition, and modulated genes related to lipid metabolism with a strong implication of peroxisome proliferator-activated receptor (PPARα)/retinoid X receptor (RXRα) involvement. In the "ongoing lipogenic state," the effect of quercetin and ferulic acid is markedly different, with fewer changes in gene expression and lipid composition, and no detectable involvement of PPARα/RXRα, with a tenfold higher concentration required to attenuate stored lipid content. CONCLUSIONS: Multiple low-dose treatment of quercetin and ferulic acid modulates lipid metabolism in adipocytes, but the effect is dramatically dependent on the metabolic state of the cell.

IL-36 Promotes Systemic IFN-I Responses in Severe Forms of Psoriasis.

Psoriasis is an immune-mediated skin disorder associated with severe systemic comorbidities. Whereas IL-36 is a key disease driver, the pathogenic role of this cytokine has mainly been investigated in skin. Thus, its effects on systemic immunity and extracutaneous disease manifestations remain poorly understood. To address this issue, we investigated the consequences of excessive IL-36 activity in circulating immune cells. We initially focused our attention on generalized pustular psoriasis (GPP), a clinical variant associated with pervasive upregulation of IL-36 signaling. By undertaking blood and neutrophil RNA sequencing, we demonstrated that affected individuals display a prominent IFN-I signature, which correlates with abnormal IL-36 activity. We then validated the association between IL-36 deregulation and IFN-I over-expression in patients with severe psoriasis vulgaris (PV). We also found that the activation of IFN-I genes was associated with extracutaneous morbidity, in both GPP and PV. Finally, we undertook mechanistic experiments, demonstrating that IL-36 acts directly on plasmacytoid dendritic cells, where it potentiates toll-like receptor (TLR)-9 activation and IFN-α production. This effect was mediated by the upregulation of PLSCR1, a phospholipid scramblase mediating endosomal TLR-9 translocation. These findings identify an IL-36/ IFN-I axis contributing to extracutaneous inflammation in psoriasis.

Long-read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel-Gruber syndrome.

The diagnostic deployment of massively parallel short-read next-generation sequencing (NGS) has greatly improved genetic test availability, speed, and diagnostic yield, particularly for rare inherited disorders. Nonetheless, diagnostic approaches based on short-read sequencing have a poor ability to accurately detect gene conversion events. We report on the genetic analysis of a family in which 3 fetuses had clinical features consistent with the autosomal recessive disorder Meckel-Gruber syndrome (MKS). Targeted NGS of 29 known MKS-associated genes revealed a heterozygous TMEM231 splice donor variant c.929+1A>G. Comparative read-depth analysis, performed to identify a second pathogenic allele, revealed an apparent heterozygous deletion of TMEM231 exon 4. To verify this result we performed single-molecule long-read sequencing of a long-range polymerase chain reaction product spanning this locus. We identified four missense variants that were absent from the short-read dataset due to the preferential mapping of variant-containing reads to a downstream TMEM231 pseudogene. Consistent with the parental segregation analysis, we demonstrate that the single-molecule long reads could be used to show that the variants are arranged in trans. Our experience shows that robust validation of apparent dosage variants remains essential to avoid the pitfalls of short-read sequencing and that new third-generation long-read sequencing technologies can already aid routine clinical care.

Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications.

The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing (NGS) strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient's father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumor cells, and for precisely identifying transgene integration sites in mouse models.

The Tudor SND1 protein is an m6A RNA reader essential for replication of Kaposi's sarcoma-associated herpesvirus.

N6-methyladenosine (m6A) is the most abundant internal RNA modification of cellular mRNAs. m6A is recognised by YTH domain-containing proteins, which selectively bind to m6A-decorated RNAs regulating their turnover and translation. Using an m6A-modified hairpin present in the Kaposi's sarcoma associated herpesvirus (KSHV) ORF50 RNA, we identified seven members from the 'Royal family' as putative m6A readers, including SND1. RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide, revealing SND1 as an m6A reader. We further demonstrate that the m6A modification of the ORF50 RNA is critical for SND1 binding, which in turn stabilises the ORF50 transcript. Importantly, SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication. This work demonstrates that members of the 'Royal family' have m6A-reading ability, greatly increasing their epigenetic functions beyond protein methylation.

Biallelic Mutations in LRRC56, Encoding a Protein Associated with Intraflagellar Transport, Cause Mucociliary Clearance and Laterality Defects.

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.

Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1).

Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1) for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles.

Cantú syndrome with coexisting familial pituitary adenoma.

CONTEXT: Pseudoacromegaly describes conditions with an acromegaly related physical appearance without abnormalities in the growth hormone (GH) axis. Acromegaloid facies, together with hypertrichosis, are typical manifestations of Cantú syndrome. CASE DESCRIPTION: We present a three-generation family with 5 affected members, with marked acromegaloid facies and prominent hypertrichosis, due to a novel missense variant in the ABCC9 gene. The proband, a 2-year-old girl, was referred due to marked hypertrichosis, noticed soon after birth, associated with coarsening of her facial appearance. Her endocrine assessment, including of the GH axis, was normal. The proband's father, paternal aunt, and half-sibling were referred to the Endocrine department for exclusion of acromegaly. Although the GH axis was normal in all, two subjects had clinically non-functioning pituitary macroadenomas, a feature which has not previously been associated with Cantú syndrome. CONCLUSIONS: Activating mutations in the ABCC9 and, less commonly, KCNJ8 genes-representing the two subunits of the ATP-sensitive potassium channel-have been linked with Cantú syndrome. Interestingly, minoxidil, a well-known ATP-sensitive potassium channel agonist, can cause a similar phenotype. There is no clear explanation why activating this channel would lead to acromegaloid features or hypertrichosis. This report raises awareness for this complex condition, especially for adult or pediatric endocrinologists who might see these patients referred for evaluation of acromegaloid features or hirsutism. The link between Cantú syndrome and pituitary adenomas is currently unclear.

Novel avian paramyxovirus isolated from gulls in Caspian seashore in Kazakhstan.

Three isolates APMV/gull/Kazakhstan/5976/2014, APMV/gull/Kazakhstan/ 5977/2014 and APMV/gull/Kazakhstan/5979/2014, were obtained from independent samples during annual surveillance for avian influenza and paramyxoviruses in wild birds from the Caspian Sea coast in Western Kazakhstan, and were initially identified as putative paramyxoviruses on the basis of electron microscopy. Hemagglutination Inhibition Assays with antisera to nine known APMV serotypes (APMV1-9) indicated no relation to any of them. Next generation sequencing of whole genome sequences indicated the three isolates were genetically identical, and had a nucleotide structure typical for all APMVs, consisting of six genes 3'-NP-P-M-F-HN-L-5'. Phylogenetic analyses, and assessment of amino acid identities, suggested the most closely related lineages to be APMV-2, 8, 10 and 15, but the novel isolate had less than 64% identity to them and all other known avian paramyxoviruses. This value was above levels considered to generally define other APMV serotypes. Estimates of the evolutionary divergence of the nucleotide sequences of the genomes of APMVs have shown that novel Kazakhstan APMV strain was closest to APMV-2, APMV-8, APMV-10 and APMV-15, with calculated distance values of 2.057, 2.058, 2.026 and 2.286 respectively, which is above values considered to differentiate other serotypes (observed minimum was 1.108 between APMV-1 and recently isolated APMV/UPO216/Korea). Together, the data suggest that isolate APMV/gull/Kazakhstan/5976/2014 and other two should be considered as the first representative of a novel APMV-20 group, and is the first time that avian paramyxoviruses have been found infecting members of the gull family, extending the known taxonomic host range.

An analysis of IL-36 signature genes and individuals with IL1RL2 knockout mutations validates IL-36 as a psoriasis therapeutic target.

Interleukin (IL)-36α, IL-36ß, and IL-36γ are innate mediators of acute epithelial inflammation. We sought to demonstrate that these cytokines are also required for the pathogenesis of plaque psoriasis, a common and chronic skin disorder, caused by abnormal T helper 17 (TH17) cell activation. To investigate this possibility, we first defined the genes that are induced by IL-36 cytokines in primary human keratinocytes. This enabled us to demonstrate a significant IL-36 signature among the transcripts that are up-regulated in plaque psoriasis and the susceptibility loci associated with the disease in genome-wide studies. Next, we investigated the impact of in vivo and ex vivo IL-36 receptor blockade using a neutralizing antibody or a recombinant antagonist. Both inhibitors had marked anti-inflammatory effects on psoriatic skin, demonstrated by statistically significant reductions in IL-17 expression, keratinocyte activation, and leukocyte infiltration. Finally, we explored the potential safety profile associated with IL-36 blockade by phenotyping 12 individuals carrying knockout mutations of the IL-36 receptor gene. We found that normal immune function was broadly preserved in these individuals, suggesting that IL-36 signaling inhibition would not substantially compromise host defenses. These observations, which integrate the results of transcriptomics and model system analysis, pave the way for early-stage clinical trials of IL-36 antagonists.

Increased Sensitivity of Diagnostic Mutation Detection by Re-analysis Incorporating Local Reassembly of Sequence Reads.

BACKGROUND: Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations. METHODS: We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases. RESULTS: ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2 × 101-bp) and NextSeq500 (2 × 151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools. CONCLUSIONS: This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.