Shariant platform: Enabling evidence sharing across Australian clinical genetic-testing laboratories to support variant interpretation.

Sharing genomic variant interpretations across laboratories promotes consistency in variant assertions. A landscape analysis of Australian clinical genetic-testing laboratories in 2017 identified that, despite the national-accreditation-body recommendations encouraging laboratories to submit genotypic data to clinical databases, fewer than 300 variants had been shared to the ClinVar public database. Consultations with Australian laboratories identified resource constraints limiting routine application of manual processes, consent issues, and differences in interpretation systems as barriers to sharing. This information was used to define key needs and solutions required to enable national sharing of variant interpretations. The Shariant platform, using both the GRCh37 and GRCh38 genome builds, was developed to enable ongoing sharing of variant interpretations and associated evidence between Australian clinical genetic-testing laboratories. Where possible, two-way automated sharing was implemented so that disruption to laboratory workflows would be minimized. Terms of use were developed through consultation and currently restrict access to Australian clinical genetic-testing laboratories. Shariant was designed to store and compare structured evidence, to promote and record resolution of inter-laboratory classification discrepancies, and to streamline the submission of variant assertions to ClinVar. As of December 2021, more than 14,000 largely prospectively curated variant records from 11 participating laboratories have been shared. Discrepant classifications have been identified for 11% (28/260) of variants submitted by more than one laboratory. We have demonstrated that co-design with clinical laboratories is vital to developing and implementing a national variant-interpretation sharing effort. This approach has improved inter-laboratory concordance and enabled opportunities to standardize interpretation practices.

Relapsing necrotising encephalomyelopathy due to RANBP2 mutation.

A young woman with a history of previously undiagnosed episodic neurological deterioration since early childhood associated with viral illnesses developed fulminant encephalitis. Standard investigations gave no diagnosis. Next-generation sequencing identified a heterozygous mutation c.1754C>T (p.Thr585Met) in the RANBP2 gene, giving the diagnosis of recurrent acute necrotising encephalopathy type 1. This condition is probably underdiagnosed, especially in adults, and should be considered in patients with recurrent encephalopathy.

Evaluation and pilot testing of a multidisciplinary model of care to mainstream genomic testing for paediatric inborn errors of immunity.

Molecular diagnosis of paediatric inborn errors of immunity (IEI) influences management decisions and alters clinical outcomes, through early use of targeted and curative therapies. The increasing demand for genetic services has resulted in growing waitlists and delayed access to vital genomic testing. To address this issue, the Queensland Paediatric Immunology and Allergy Service, Australia, developed and evaluated a mainstreaming model of care to support point-of-care genomic testing for paediatric IEI. Key features of the model of care included a genetic counsellor embedded in the department, state-wide multidisciplinary team meetings, and variant prioritisation meetings to review whole exome sequencing (WES) data. Of the 62 children presented at the MDT, 43 proceeded to WES, of which nine (21%) received a confirmed molecular diagnosis. Changes to treatment and management were reported for all children with a positive result, including curative hematopoietic stem cell transplantation (n = 4). Four children were also referred for further investigations of variants of uncertain significance or additional testing due to ongoing suspicion of genetic cause after negative result. Demonstrating engagement with the model of care, 45% of the patients were from regional areas and on average, 14 healthcare providers attended the state-wide multidisciplinary team meetings. Parents demonstrated understanding of the implications of testing, reported minimal decisional regret post-test, and identified benefits to genomic testing. Overall, our program demonstrated the feasibility of a mainstreaming model of care for paediatric IEI, improved access to genomic testing, facilitated treatment decision-making, and was acceptable to parents and clinicians alike.

A Multi-Disciplinary Team Approach to Genomic Testing for Drug-Resistant Epilepsy Patients-The GENIE Study.

BACKGROUND: The genomic era has led to enormous progress in clinical care and a multi-disciplinary team (MDT) approach is imperative for integration of genomics into epilepsy patient care. METHODS: The MDT approach involved patient selection, genomic testing choice, variant discussions and return of results. Genomics analysis included cytogenomic testing and whole exome sequencing (WES). Neurologist surveys were undertaken at baseline and after genomic testing to determine if genomic diagnoses would alter their management, and if there was a change in confidence in genomic testing and neurologist perceptions of the MDT approach. RESULTS: The total diagnostic yield from all genomic testing was 17% (11/66), with four diagnoses from cytogenomic analyses. All chromosomal microarray (CMA) diagnoses were in patients seen by adult neurologists. Diagnostic yield for WES was 11% (7/62). The most common gene with pathogenic variants was DCX, reported in three patients, of which two were mosaic. The genomic diagnosis impacted management in 82% (9/11). There was increased confidence with integrating genomics into clinical care (Pearson chi square = 83, p = 0.004) and qualitative comments were highly supportive of the MDT approach. CONCLUSIONS: We demonstrated diagnostic yield from genomic testing, and the impact on management in a cohort with drug-resistant epilepsy. The MDT approach increased confidence in genomic testing and neurologists valued the input from this approach. The utility of CMA was demonstrated in epilepsy patients seen by adult neurologists as was the importance of considering mosaicism for previously undiagnosed patients.

Selective induction of the Notch ligand Jagged-1 in macrophages by soluble egg antigen from Schistosoma mansoni involves ERK signalling.

Soluble egg antigen (SEA) from the helminth Schistosoma mansoni promotes T helper type 2 (Th2) responses by modulating antigen-presenting cell function. The Jagged/Notch pathway has recently been implicated in driving Th2 development. We show here that SEA rapidly up-regulated mRNA and protein expression of the Notch ligand Jagged-1 in both murine bone marrow-derived macrophages (BMMs) and human monocyte-derived macrophages (HMDMs). Another potential Th2-promoting factor, interleukin (IL)-33, was not transcriptionally induced by SEA in BMMs. Up-regulation of Jagged-1 mRNA by SEA was also apparent in conventional dendritic cells (DCs), although the effect was less striking than in BMMs. Conversely, SEA-pulsed DCs, but not BMMs, promoted IL-4 production upon T-cell activation, suggesting that Jagged-1 induction alone is insufficient for instructing Th2 development. A comparison of the responses initiated in BMMs by SEA and the bacterial endotoxin lipopolysaccharide (LPS) revealed common activation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and p38 phosphorylation, as well as induction of Jagged-1 mRNA. However, only LPS triggered IkappaB degradation, phosphorylation of c-Jun N-terminal kinase (Jnk) and signal transducer and activator of transcription 1 (Stat1) Tyr701, and IL-33 and IL-12p40 mRNA up-regulation. Inducible gene expression was modified by the presence of the macrophage growth factor colony-stimulating factor (CSF)-1, which inhibited Jagged-1 induction by SEA and LPS, but enhanced LPS-induced IL-12p40 expression. Unlike LPS, SEA robustly activated signalling in HEK293 cells expressing either Toll-like receptor 2 (TLR2) or TLR4/MD2. Pharmacological inhibition of the ERK-1/2 pathway impaired SEA- and LPS-inducible Jagged-1 expression in BMMs. Taken together, our data suggest that Jagged-1 is an ERK-dependent target of TLR signalling that has a macrophage-specific function in the response to SEA.

PU.1 and ICSBP control constitutive and IFN-gamma-regulated Tlr9 gene expression in mouse macrophages.

Macrophages are activated by unmethylated CpG-containing DNA (CpG DNA) via TLR9. IFN-gamma and LPS can synergize with CpG DNA to enhance proinflammatory responses in murine macrophages. Here, we show that LPS and IFN-gamma up-regulated Tlr9 mRNA in murine bone marrow-derived macrophages (BMM). The ability of LPS and IFN-gamma to induce Tlr9 mRNA expression in BMM was dependent on the presence of the growth factor, CSF-1, which is constitutively present in vivo. However, there were clear differences in mechanisms of Tlr9 mRNA induction. LPS stimulation rapidly removed the CSF-1 receptor (CSF-1R) from the cell surface, thereby blocking CSF-1-mediated transcriptional repression and indirectly inducing Tlr9 mRNA expression. By contrast, IFN-gamma activated the Tlr9 promoter directly and only marginally affected cell surface CSF-1R expression. An approximately 100-bp proximal promoter of the murine Tlr9 gene was sufficient to confer basal and IFN-gamma-inducible expression in RAW264.7 cells. A composite IFN regulatory factor (IRF)/PU.1 site upon the major transcription start site was identified. Mutation of the binding sites for PU.1 or IRF impaired basal promoter activity, but only the IRF-binding site was required for IFN-gamma induction. The mRNA expression of the IRF family member IFN consensus-binding protein [(ICSBP)/IRF8] was coregulated with Tlr9 in macrophages, and constitutive and IFN-gamma-inducible Tlr9 mRNA expression was reduced in ICSBP-deficient BMM. This study therefore characterizes the regulation of mouse Tlr9 expression and defines a molecular mechanism by which IFN-gamma amplifies mouse macrophage responses to CpG DNA.

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression.

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.

LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action.

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-1R rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK1/2 phosphorylation. Using toll-like receptor (tlr)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R.

CpG DNA activates survival in murine macrophages through TLR9 and the phosphatidylinositol 3-kinase-Akt pathway.

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-alpha production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.