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Infections are a major contributor to lupus disease. Uropathogenic E. coli (UPEC) is responsible for the majority of urinary tract infections in both healthy individuals and lupus patients. We have previously demonstrated that bacterial amyloid curli complexes of curli/DNA, produced by E.coli, can accelerate disease in mouse models of lupus. Moreover we have extended these findings to human lupus and demonstrate that curli/DNA complexes mimic lupus autoantigens and that patients with chronic bacteriuria and high levels of anti-curli/DNA have higher levels of anti-dsDNA, more flares and a proinflammatory profile. These findings suggest that curli/DNA complexes and subclinical chronic urinary bacterial infections might be a trigger and a propagator of autoimmunity via activation of the innate and adaptive immune system. Based on our previous results, we hypothesize that exposure to UPEC containing curli/eDNA complexes could also activate neutrophils, the first responders to bacterial infections, and specifically via generation of neutrophil extracellular traps (NETs), a fundamental mechanism to clear bacteria and a recently appreciated pathogenic mechanism in lupus. Neutrophil extracellular traps (NETs) are part of the innate immune system and are pathogenic in SLE. We therefore investigated 56 lupus patients who met at least 4 SLICC criteria. Results were compared to 20 age, sex, and race matched healthy controls. We found that curli/eDNA induced more NETs in SLE PMNs compared to healthy controls. In SLE, patients who were high inducers of NETs triggered by curli/eDNA complexes were also a high inducer of NETs triggered by LPS and PMA. Interestingly, patients who were anti- dsDNA positive made more NETs in response to curli/eDNA complexes. Moreover, we found patients who are anti-dsDNA positive responded highly to curli/eDNA complexes and LPS. We did not observe this in patients who were anti-dsDNA negative. Mechanistically, we found that curli/eDNA induce NETs via NADPH oxidase. Finally, we found patients who had bacteriuria had a higher amount of NET production in response to curli/eDNA complexes and PMA compared to patients with no bacteriuria. We conclude1) that lupus PMNs are in a chronic inflammatory state. And 2) that curli/eDNA complexes can activate neutrophils and exposure to UPECs could be a mechanism to sustain autoantigens in the form of neutrophil extracellular traps.

BackgroundInfections are a major contributor to lupus disease. We have previously demonstrated that bacterial amyloid curli, produced by E.coli, can accelerate disease in mouse models of lupus. Interestingly curli incorporates extracellular DNA, which in turn can be both adjuvant and a self-antigen in lupus. Uropathogenic E. coli (UPEC) is responsible for the majority of urinary tract infections in SLE. Based on our previous results, we hypothesize that exposure to UPEC triggers anti-curli/eDNA antibodies and curli/eDNA complexes can trigger the innate immune system.MethodsWe investigated 98 lupus patients who met at least 4 SLICC criteria. Results were compared to 54 age, sex and race matched healthy controls. We tested the production of anti-curli/DNA complex for both IgG and IgA subclasses. We than correlated the levels of anti-curli/DNA antibodies with clinical parameters. Finally, we treated human neutrophils with curli/eDNA complexes.ResultsWe found that curli/eDNA induces neutrophil extracellular traps in a ROS-dependent manner. Anti-curli/eDNA IgG levels were detected in lupus and controls plasma and the levels correlated with persistent bacteriuria (p<0.05) and disease flares in lupus patients. In addition, anti-dsDNA could bind to anti-curli/eDNA complexes.ConclusionsWe conclude curli/eDNA complexes can activate the innate and adaptive immune system and could be a mechanism to sustain disease in lupus.AcknowledgmentsWe thank Drs. Marc Monestier and Philip Cohen for their insightful suggestions. We also thank the generosity of the lupus patients from the Temple Lupus Program.

Purpose: Multiplex genetic testing, including both moderate- and high-penetrance genes for cancer susceptibility, is associated with greater uncertainty than traditional testing, presenting challenges to informed consent and genetic counseling. We sought to develop a new model for informed consent and genetic counseling for four ongoing studies. Methods: Drawing from professional guidelines, literature, conceptual frameworks, and clinical experience, a multidisciplinary group developed a tiered-binned genetic counseling approach proposed to facilitate informed consent and improve outcomes of cancer susceptibility multiplex testing. Results: In this model, tier 1 “indispensable” information is presented to all patients. More specific tier 2 information is provided to support variable informational needs among diverse patient populations. Clinically relevant information is “binned” into groups to minimize information overload, support informed decision making, and facilitate adaptive responses to testing. Seven essential elements of informed consent are provided to address the unique limitations, risks, and uncertainties of multiplex testing. Conclusion: A tiered-binned model for informed consent and genetic counseling has the potential to address the challenges of multiplex testing for cancer susceptibility and to support informed decision making and adaptive responses to testing. Future prospective studies including patient-reported outcomes are needed to inform how to best incorporate multiplex testing for cancer susceptibility into clinical practice. Genet Med 17 6, 485–492.

Population-based cancer registries (PBCRs) generate measures of cancer incidence and survival that are essential for cancer surveillance, research, and cancer control strategies. In 2014, the Toronto Paediatric Cancer Stage Guidelines were developed to standardise how PBCRs collect data on the stage at diagnosis for childhood cancer cases. These guidelines have been implemented in multiple jurisdictions worldwide to facilitate international comparative studies of incidence and outcome. Robust stratification by risk also requires data on key non-stage prognosticators (NSPs). Key experts and stakeholders used a modified Delphi approach to establish principles guiding paediatric cancer NSP data collection. With the use of these principles, recommendations were made on which NSPs should be collected for the major malignancies in children. The 2014 Toronto Stage Guidelines were also reviewed and updated where necessary. Wide adoption of the resultant Paediatric NSP Guidelines and updated Toronto Stage Guidelines will enhance the harmonisation and use of childhood cancer data provided by PBCRs.

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene1. The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches2–5. For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases6–8. This includes muscle biopsies from patients with undiagnosed rare muscle disorders6,9, and cultured fibroblasts from patients with mitochondrial disorders7. However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.A diagnostic tool based on blood RNA-seq is shown to identify causal genes and variants linked to clinical phenotypes in individuals with rare diseases for which whole-exome genetic sequencing was uninformative.

Background Short tandem repeats (STRs) are widely distributed across the human genome and are associated with numerous neurological disorders. However, the extent that STRs contribute to disease is likely under-estimated because of the challenges calling these variants in short read next generation sequencing data. Several computational tools have been developed for STR variant calling, but none fully address all of the complexities associated with this variant class. Results Here we introduce LUSTR which is designed to address some of the challenges associated with STR variant calling by enabling more flexibility in defining STR loci, allowing for customizable modules to tailor analyses, and expanding the capability to call somatic and multiallelic STR variants. LUSTR is a user-friendly and easily customizable tool for targeted or unbiased genome-wide STR variant screening that can use either predefined or novel genome builds. Using both simulated and real data sets, we demonstrated that LUSTR accurately infers germline and somatic STR expansions in individuals with and without diseases. Conclusions LUSTR offers a powerful and user-friendly approach that allows for the identification of STR variants and can facilitate more comprehensive studies evaluating the role of pathogenic STR variants across human diseases.

The “diagnostic odyssey” describes the process those with undiagnosed conditions undergo to identify a diagnosis. Throughout this process, families of children with undiagnosed conditions have multiple opportunities to decide whether to continue or stop their search for a diagnosis and accept the lack of a diagnostic label. Previous studies identified factors motivating a family to begin searching, but there is limited information about the decision-making process in a prolonged search and how the affected child impacts a family’s decision. This study aimed to understand how families of children with undiagnosed diseases decide whether to continue to pursue a diagnosis after standard clinical testing has failed. Parents who applied to the Undiagnosed Disease Network (UDN) at the National Institutes of Health (NIH) were recruited to participate in semi-structured interviews. The 2015 Supportive Care Needs model by Pelenstov, which defines critical needs in families with rare/undiagnosed diseases, provided a framework for interview guide development and transcript analysis (Pelentsov et al in Disabil Health J 8(4):475–491, 2015. https://doi.org/10.1016/J.DHJO.2015.03.009 ). A deductive, iterative coding approach was used to identify common unifying themes. Fourteen parents from 13 families were interviewed. The average child’s age was 11 years (range 3–18) and an average 63% of their life had been spent searching for a diagnosis. Our analysis found that alignment or misalignment of parent and child needs impact the trajectory of the diagnostic search. When needs and desires align, reevaluation of a decision to pursue a diagnosis is limited. However, when there is conflict between parent and child desires, there is reevaluation, and often a pause, in the search. This tension is exacerbated when children are adolescents and attempting to balance their dependence on parents for medical care with a natural desire for independence. Our results provide novel insights into the roles of adolescents in the diagnostic odyssey. The tension between desired and realistic developmental outcomes for parents and adolescents impacts if, and how, the search for a diagnosis progresses.

Background When applying genomic medicine to a rare disease patient, the primary goal is to identify one or more genomic variants that may explain the patient’s phenotypes. Typically, this is done through annotation, filtering, and then prioritization of variants for manual curation. However, prioritization of variants in rare disease patients remains a challenging task due to the high degree of variability in phenotype presentation and molecular source of disease. Thus, methods that can identify and/or prioritize variants to be clinically reported in the presence of such variability are of critical importance. Methods We tested the application of classification algorithms that ingest variant annotations along with phenotype information for predicting whether a variant will ultimately be clinically reported and returned to a patient. To test the classifiers, we performed a retrospective study on variants that were clinically reported to 237 patients in the Undiagnosed Diseases Network. Results We treated the classifiers as variant prioritization systems and compared them to four variant prioritization algorithms and two single-measure controls. We showed that the trained classifiers outperformed all other tested methods with the best classifiers ranking 72% of all reported variants and 94% of reported pathogenic variants in the top 20. Conclusions We demonstrated how freely available binary classification algorithms can be used to prioritize variants even in the presence of real-world variability. Furthermore, these classifiers outperformed all other tested methods, suggesting that they may be well suited for working with real rare disease patient datasets.

Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease–gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein–protein association neighbors. Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease–gene associations and ranked the correct seeded variant in up to 87% when detectable disease–gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders.

Objective ACTN2, encoding alpha‐actinin‐2, is essential for cardiac and skeletal muscle sarcomeric function. ACTN2 variants are a known cause of cardiomyopathy without skeletal muscle involvement. Recently, specific dominant monoallelic variants were reported as a rare cause of core myopathy of variable clinical onset, although the pathomechanism remains to be elucidated. The possibility of a recessively inherited ACTN2‐myopathy has also been proposed in a single series. Methods We provide clinical, imaging, and histological characterization of a series of patients with a novel biallelic ACTN2 variant. Results We report seven patients from five families with a recurring biallelic variant in ACTN2: c.1516A>G (p.Arg506Gly), all manifesting with a consistent phenotype of asymmetric, progressive, proximal, and distal lower extremity predominant muscle weakness. None of the patients have cardiomyopathy or respiratory insufficiency. Notably, all patients report Palestinian ethnicity, suggesting a possible founder ACTN2 variant, which was confirmed through haplotype analysis in two families. Muscle biopsies reveal an underlying myopathic process with disruption of the intermyofibrillar architecture, Type I fiber predominance and atrophy. MRI of the lower extremities demonstrate a distinct pattern of asymmetric muscle involvement with selective involvement of the hamstrings and adductors in the thigh, and anterior tibial group and soleus in the lower leg. Using an in vitro splicing assay, we show that c.1516A>G ACTN2 does not impair normal splicing. Interpretation This series further establishes ACTN2 as a muscle disease gene, now also including variants with a recessive inheritance mode, and expands the clinical spectrum of actinopathies to adult‐onset progressive muscle disease.