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OBJECTIVE: WWOX is an autosomal recessive cause of early infantile developmental and epileptic encephalopathy (WWOX-DEE), also known as WOREE (WWOX-related epileptic encephalopathy). We analyzed the epileptology and imaging features of WWOX-DEE, and investigated genotype-phenotype correlations, particularly with regard to survival. METHODS: We studied 13 patients from 12 families with WWOX-DEE. Information regarding seizure semiology, comorbidities, facial dysmorphisms, and disease outcome were collected. Electroencephalographic (EEG) and brain magnetic resonance imaging (MRI) data were analyzed. Pathogenic WWOX variants from our cohort and the literature were coded as either null or missense, allowing individuals to be classified into one of three genotype classes: (1) null/null, (2) null/missense, (3) missense/missense. Differences in survival outcome were estimated using the Kaplan-Meier method. RESULTS: All patients experienced multiple seizure types (median onset = 5 weeks, range = 1 day-10 months), the most frequent being focal (85%), epileptic spasms (77%), and tonic seizures (69%). Ictal EEG recordings in six of 13 patients showed tonic (n = 5), myoclonic (n = 2), epileptic spasms (n = 2), focal (n = 1), and migrating focal (n = 1) seizures. Interictal EEGs demonstrated slow background activity with multifocal discharges, predominantly over frontal or temporo-occipital regions. Eleven of 13 patients had a movement disorder, most frequently dystonia. Brain MRIs revealed severe frontotemporal, hippocampal, and optic atrophy, thin corpus callosum, and white matter signal abnormalities. Pathogenic variants were located throughout WWOX and comprised both missense and null changes including five copy number variants (four deletions, one duplication). Survival analyses showed that patients with two null variants are at higher mortality risk (p-value = .0085, log-rank test). SIGNIFICANCE: Biallelic WWOX pathogenic variants cause an early infantile developmental and

In scaling up an ultra-rapid genomics program, we used implementation science principles to design and investigate influences on implementation and identify strategies required for sustainable "real-world" services. Interviews with key professionals revealed the importance of networks and relationship building, leadership, culture, and the relative advantage afforded by ultra-rapid genomics in the care of critically ill children. Although clinical geneticists focused on intervention characteristics and the fit with patient-centered care, intensivists emphasized the importance of access to knowledge, in particular from clinical geneticists. The relative advantage of ultra-rapid genomics and trust in consistent and transparent delivery were significant in creating engagement at initial implementation, with appropriate resourcing highlighted as important for longer term sustainability of implementation. Our findings demonstrate where common approaches can be used and, significantly, where there is a need to tailor support by professional role and implementation phase, to maximize the potential of ultra-rapid genomic testing to improve patient care.

Introduction: Implementation of rapid genomic testing in neonatal and paediatric intensive care units (NICUs/PICUs) is gathering momentum, and requires the development of systems capable of consistent delivery across multi-site networks. Method(s): We developed a rapid genomic diagnosis program involving 10 Australian hospitals and two laboratories with the aim of providing test results in <5 days for acutely unwell paediatric patients with suspected monogenic disorders. Rapid exome sequencing (rES) was performed as trios when possible, and analysis utilised multidisciplinary expertise. Experience was shared between clinical sites, laboratories, and professional groups to enable collective learning. Result(s): The program considered 123 patients for rES over 10 months, and approved 114 (93%). Five families declined testing (4.4%), and nine (7.9%) were withdrawn due to change in clinical circumstances. Of 100 patients tested, 51 received a diagnosis. Eleven of the diagnoses (21%) were made using approaches augmenting standard ES analysis: mitochondrial genome sequencing, ES-based copy number analysis, matchmaking of emerging genes, reverse phenotyping and RNA analysis. Median time from hospital admission to consent was 6 days (range 0-64 days); median time from sample receipt to clinical ES report was 3 days (range 2-7 days). The total cost of testing was AU$1,123,000/701,638 (AU$11,230/7,016 per case). Changes in management following a result occurred in 77% of diagnosed patients and 10% of undiagnosed patients. Conclusion(s): We demonstrate the feasibility of a national, highly integrated clinical-laboratory approach to rapid genomic diagnosis, which delivers results within a timeframe relevant to acute paediatrics, while optimising clinical utility and resource allocation.

Multiple studies have shown that genomic testing has a high diagnostic yield and an impact on clinical management for patients with suspected genetic conditions. Therefore, there has been a push worldwide to apply rapid genomic sequencing in critically ill neonatal and pediatric patients. The goal of this study was to investigate the practicality of applying ultrarapid genomic testing for critically ill neonatal and pediatric patients with suspected monogenic conditions in Australia. The study recruited a total of 108 patients prospectively from March 2018 to February 2019, and data were collected until May 2019. Of the 12 hospitals that acted as collaborating sites, 5 were women's hospitals, 3 women's and children's hospitals, and 4 children's hospitals. Eligible patients included those who were admitted to a neonatal or pediatric intensive care unit (NICU or PICU) and were referred to clinical genetics for a possible monogenic condition. Chromosomal microarray was a requirement before enrollment if there was a suspected chromosomal condition. Chromosomal microarrays were performed concurrently with ultrarapid exome sequencing when the probability of a positive result on microarray was low. Additionally, rapid mitochondrial genome sequencing was performed alongside ultrarapid exome sequencing if a mitochondrial condition was suspected. When possible, ultrarapid exome sequencing was performed in both parents as well as the child (trio). The primary outcome of this study measured the time from the last sample received to the ultrarapid exome sequencing report finalized. Other outcomes measured were the diagnostic yield, change in clinical management after the report was finalized, number of reports returned before hospital discharge, and time from admission to the report being finalized. Of the 108 patients enrolled, the median age was 28 days, with a range of 0 days to 17 years. Overall, 34% were female, 57% were NICU admissions, 33% were PICU admissions, and 9% wer

Purpose: To explore parental experiences of ultrarapid genomic testing for their critically unwell infants and children. Method(s): Parents of critically unwell children who participated in a national ultrarapid genomic diagnosis program were surveyed >12 weeks after genomic results return. Surveys consisted of custom questions and validated scales, including the Decision Regret Scale and Genomics Outcome Scale. Result(s): With 96 survey invitations sent, the response rate was 57% (n = 55). Most parents reported receiving enough information during pretest (n = 50, 94%) and post-test (n = 44, 83%) counseling. Perceptions varied regarding benefits of testing, however most parents reported no or mild decision regret (n = 45, 82%). The majority of parents (31/52, 60%) were extremely concerned about the condition recurring in future children, regardless of actual or perceived recurrence risk. Parents whose child received a diagnostic result reported higher empowerment. Conclusion(s): This study provides valuable insight into parental experiences of ultrarapid genomic testing in critically unwell children, including decision regret, empowerment, and post-test reproductive planning, to inform design and delivery of rapid diagnosis programs. The findings suggest considerations for pre- and post-test counseling that may influence parental experiences during the testing process and beyond, such as the importance of realistically conveying the likelihood for clinical and/or personal utility.Copyright © 2020, American College of Medical Genetics and Genomics.

Background: Emerging evidence that rapid turnaround times impact the clinical utility of genomic testing in acute paediatrics is driving widespread adoption. However, little is known about the experience that parents of critically unwell infants and children have during the testing process and beyond. Method(s): Participants were recruited as part of the Australian Genomics Acute Care study, a national rapid genomic diagnosis program for infants and children admitted to intensive care with suspected genetic conditions. Pre-and post-test counselling was provided by genetic health professionals. Over 95% of parents offered testing gave consent. Results were available within five days of sample receipt. Parents were surveyed >12 weeks after results return. We explored parental experiences with consent processes, perceived impact of testing on child health, relationships and reproductive decisions. This questionnaire included the Decision Regret, Short Form Genetic Counselling Outcomes and PedsQL Family Impact Module scales. Result(s): From 21 respondents in the first six months (RR = 54%), most felt they received enough information during pre-test (n = 21, 100%) and post-test (n = 18, 86%) counselling. No respondents reported decisional regret regarding testing. Perceptions varied about the benefits of rapid genomic sequencing for the child. The majority of respondents (n = 13, 62%) were extremely concerned about the condition occurring in future children, regardless of their actual or self-perceived recurrence risk. Eight respondents (38%) reported the test impacted their reproductive plans. Importance: Understanding parental experiences, opinions, and the short and long term impacts on families will guide the design and delivery of rapid genomic diagnosis programs.

Importance: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems. Objective(s): To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system. Design, Setting, and Participant(s): Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination, distributed leadership, and collective learning was used to facilitate adoption. Exposures: Ultra-rapid exome sequencing. Main Outcomes and Measures: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge. Result(s): The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The mean time from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI, 3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

Background/Aim: Implementation of rapid genomic testing in neonatal and pediatric intensive care units (NICUs/PICUs) is gathering momentum, and requires the development of systems capable of consistent delivery across multiple sites. Method(s): We developed a rapid genomic diagnosis program involving 10 Australian hospitals and two laboratories with the aim of providing test results in <5 days for acutely unwell pediatric patients with suspected monogenic disorders. Rapid exome sequencing (rES) was performed as trios when possible, and analysis utilized multidisciplinary expertise. Experience was shared between clinical sites, laboratories, and professional groups to enable collective learning. Result(s): The program considered 123 patients for rES over 10 months, and approved 114 (93%). Five families declined testing (4.4%), and nine (7.9%) were withdrawn due to change in clinical circumstances. Of 100 patients tested, 53 received a diagnosis. Twelve of the diagnoses (23%) were made using approaches augmenting standard ES analysis: mitochondrial genome sequencing, ES-based copy number analysis, matchmaking of emerging genes, reverse phenotyping and RNA analysis. Median time from hospital admission to consent was 6 days (range 0-64 days); median time from sample receipt to clinical ES report was 3 days (range 2-7 days). The total cost of testing was AU $1,123,000 (AU$11,230 per case). Changesin management following a result occurred in 77% of diagnosed patients and 10% of undiag-nosed patients. Conclusion(s): We demonstrate the feasibility of a national, highly integrated clinical-laboratory approach to rapid genomic diagnosis, which delivers results within a timeframe relevant to acute pediatrics, while optimizing clinical utility and resource allocation.

Background: Emerging evidence that rapid turnaround times impact the clinical utility of genomic testing in acute pediatrics is driving widespread adoption. However, little is known about the experience that parents of critically unwell infants and children have during the testing process and beyond. Method(s): Participants were recruited as part of the Australian Genomics Acute Care study, a national rapid genomic diagnosis program for infants and children admitted to intensive care with suspected genetic conditions. Pre- and post-test counseling was provided by genetic health professionals. Over 95% of parents offered testing gave consent. Results were available within 5 days of sample receipt. Parents were surveyed >12 weeks after results return. We explored parental experiences with consent processes, perceived impact of testing on child health, relationships and reproductive decisions. This questionnaire included the Decision Regret, Short Form Genetic Counselling Outcomes and PedsQL Family Impact Module scales. Result(s): From 21 respondents in the first 6 months (RR = 54%), most felt they received enough information during pre-test (n = 21, 100%) and post-test (n = 18, 86%) counseling. No respondents reported decisional regret regarding testing. Perceptions varied about the benefits of rapid genomic sequencing for the child. The majority of respondents (n = 13, 62%) were extremely concerned about the condition occurring in future children, regardless of their actual or self-perceived recurrence risk. Eight respondents (38%) reported the test impacted their reproductive plans. Importance: Understanding parental experiences, opinions, and the short and long term impacts on families will guide the design and delivery of rapid genomic diagnosis programs.

Purpose: Whole exome sequencing (WES) is rapidly becoming the standard of care for genetic services. We present the results of 500 clinical exomes performed at the Randwick Genetic Laboratory. Method(s): WES was performed in 204 probands with suspected Mendelian disorders, together with family members. Ampliseq RDY exome, libraries were analyzed on a Life Technologies Proton instrument. Data were analyzed using an in house pipeline with variant reporting following ACMG guidelines. Result(s): WES resulted in greater than 43% definitive findings with a small number of variants of uncertain significance. The diagnostic rate has increased over time, likely reflecting refinements in clinician referral practice as well as improved functionality of bioinformatics pipelines. Two families (1%)with Cantu and MoyaMoya syndromes had results which could lead to pharmacologic interventions. 33 families (16%) had De novo variants and seven (3.5%) were X-linked with significant implications for recurrence risk. Two prenatal diagnoses have been performed based on WES results. A diagnosis was made in 6/8 (75%) rapid turnaround studies, including two during pregnancy. Almost 10% of the diagnoses were unanticipated by the clinical teams and involved significant changes in diagnostic category. Four likely novel genes involved in known biological pathways were identified, enabling research participation. Conclusion(s): The minimum number of management changing outcomes was at least 20% within the 12 month period of this study. The number of secondary findings was small at about 1%. This demonstrates that WES is characterized by high levels of patient safety and clinical utility with likely cost effectiveness.

Background: Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with a population frequency of approximately 1 in 10,000. The most common epigenetic defect in BWS is a loss of methylation (LOM) at the 11p15.5 imprinting centre, KCNQ1OT1 TSS-DMR, and affects 50% of cases. We hypothesised that genetic factors linked to folate metabolism may play a role in BWS predisposition via effects on methylation maintenance at KCNQ1OT1 TSS-DMR. Result(s): Single nucleotide variants (SNVs) in the folate pathway affecting methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR), cystathionine beta-synthase (CBS) and methionine adenosyltransferase (MAT1A) were examined in 55 BWS patients with KCNQ1OT1 TSS-DMR LOM and in 100 unaffected cases. MTHFR rs1801133: C>T was more prevalent in BWS with KCNQ1OT1 TSS-DMR LOM (p<0.017); however, the relationship was not significant when the Bonferroni correction for multiple testing was applied (significance, p=0.0036). None of the remaining 13 SNVs were significantly different in the two populations tested. The DNMT1 locus was screened in 53 BWS cases, and three rare missense variants were identified in each of three patients: rs138841970: C>T, rs150331990: A>G and rs757460628: G>A encoding NP_001124295 p.Arg136Cys, p.His1118Arg and p.Arg1223His, respectively. These variants have population frequencies of less than 1 in 1000 and were absent from 100 control cases. Functional characterization using a hemimethylated DNA trapping assay revealed a reduced methyltransferase activity relative to wild-type DNMT1 for each variant ranging from 40 to 70% reduction in activity. Conclusion(s): This study is the first to examine folate pathway genetics in BWS and to identify rare DNMT1 missense variants in affected individuals. Our data suggests that reduced DNMT1 activity could affect maintenance of methylation at KCNQ1OT1 TSS-DMR in some cases of BWS, possibl

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with a population frequency of approximately 1 in 10,000. The most common epigenetic defect in BWS is a loss of methylation (LOM) at the 11p15.5 imprinting centre, KCNQ1OT1 TSS-DMR, and affects 50% of cases. We hypothesised that genetic factors linked to folate metabolism may play a role in BWS predisposition via effects on methylation maintenance at KCNQ1OT1 TSS-DMR. RESULTS: Single nucleotide variants (SNVs) in the folate pathway affecting methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR), cystathionine beta-synthase (CBS) and methionine adenosyltransferase (MAT1A) were examined in 55 BWS patients with KCNQ1OT1 TSS-DMR LOM and in 100 unaffected cases. MTHFR rs1801133: C>T was more prevalent in BWS with KCNQ1OT1 TSS-DMR LOM (p < 0.017); however, the relationship was not significant when the Bonferroni correction for multiple testing was applied (significance, p = 0.0036). None of the remaining 13 SNVs were significantly different in the two populations tested. The DNMT1 locus was screened in 53 BWS cases, and three rare missense variants were identified in each of three patients: rs138841970: C>T, rs150331990: A>G and rs757460628: G>A encoding NP_001124295 p.Arg136Cys, p.His1118Arg and p.Arg1223His, respectively. These variants have population frequencies of less than 1 in 1000 and were absent from 100 control cases. Functional characterization using a hemimethylated DNA trapping assay revealed a reduced methyltransferase activity relative to wild-type DNMT1 for each variant ranging from 40 to 70% reduction in activity. CONCLUSIONS: This study is the first to examine folate pathway genetics in BWS and to identify rare DNMT1 missense variants in affected individuals. Our data suggests that reduced DNMT1 activity could affect maintenance of methylation at KCNQ1OT1 TSS-DMR in some cases of BWS, possibl

Contains fulltext : 172588.pdf (publisher's version ) (Open Access), Prominent skin involvement is a defining characteristic of autoinflammatory disorders caused by abnormal IL-1 signaling. However, the pathways and cell types that drive cutaneous autoinflammatory features remain poorly understood. We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a model of autoinflammatory disorders with predominant cutaneous manifestations. We specifically characterized the impact of mutations affecting AP1S3, a disease gene previously identified by our group and validated here in a newly ascertained patient resource. We first showed that AP1S3 expression is distinctively elevated in keratinocytes. Because AP1S3 encodes a protein implicated in autophagosome formation, we next investigated the effects of gene silencing on this pathway. We found that AP1S3 knockout disrupts keratinocyte autophagy, causing abnormal accumulation of p62, an adaptor protein mediating NF-kappaB activation. We showed that as a consequence, AP1S3-deficient cells up-regulate IL-1 signaling and overexpress IL-36alpha, a cytokine that is emerging as an important mediator of skin inflammation. These abnormal immune profiles were recapitulated by pharmacological inhibition of autophagy and verified in patient keratinocytes, where they were reversed by IL-36 blockade. These findings show that keratinocytes play a key role in skin autoinflammation and identify autophagy modulation of IL-36 signaling as a therapeutic target.

Contains fulltext : 168023.pdf (publisher's version ) (Closed access), Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

An unanticipated and tremendous amount of the noncoding sequence of the human genome is transcribed. Long noncoding RNAs (IncRNAs) constitute a significant fraction of non-protein-coding transcripts; however, their functions remain enigmatic. We demonstrate that deletions of a small noncoding differentially methylated region at 16q24.1, including IncRNA genes, cause a lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), with parent-of-origin effects. We identify overlapping deletions 250 kb upstream of FOXF1 in nine patients with ACD/MPV that arose de novo specifically on the maternally inherited chromosome and delete lung-specific IncRNAgenes. These deletions define a distant cis-regulatory region that harbors, besides lncRNAgenes, also a differentially methylated CpGisland, binds GLI2 depending on the methylation status of this CpG island, and physically interacts with and up-regulates the FOXF1 promoter. Wesuggest that lung-transcribed 16q24.1 IncRNAs may contribute to long-range regulation of FOXF1 by GLI2 and other transcription factors. Perturbation of IncRNA-mediated chromatin interactions may, in general, be responsible for position effect phenomena and potentially cause many disorders of human development.

This report forms part of a series on the UK component of the Comparative Nonprofit Sector Project, a thirteen-country study of the voluntary or non-profit sector. The international study was initiated by the Institute for Policy Studies at Johns Hopkins University, Baltimore, under the direction of Lester Salamon and Helmut Anheier. The other twelve countries are Brazil, Egypt, France, Germany, Ghana, Hungary, India, Italy, Japan, Sweden, Thailand and the USA. The full UK study contains a number of local or national surveys, including that which is reported here. The full UK results will be reported in Kendall and Knapp, 1994. The report is laid out in two sections, the first focusing on a national survey of voluntary organisations in mental health in the United Kingdom, with results based entirely upon the data collected by this survey, and the second combining these data with other sources to extrapolate to national levels of finance and staffing levels for the whole of this sector including organisations with both local and national catchment areas. DefinitionsThe nonprofit sector is defined as comprising organisations which are formally constituted, independent of government, not distributing profits to those who control them, self-goveming, primarily non-sacramental, not party political and benefitting to a meaningful degree from voluntarism. These criteria are arguably enshrined in UK charity law but also describe many organisations which do not hold charity status.