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Critically ill infants and children with rare diseases need equitable access to rapid and accurate diagnosis to direct clinical management. Over 2 years, the Acute Care Genomics program provided whole-genome sequencing to 290 families whose critically ill infants and children were admitted to hospitals throughout Australia with suspected genetic conditions. The average time to result was 2.9 d and diagnostic yield was 47%. We performed additional bioinformatic analyses and transcriptome sequencing in all patients who remained undiagnosed. Long-read sequencing and functional assays, ranging from clinically accredited enzyme analysis to bespoke quantitative proteomics, were deployed in selected cases. This resulted in an additional 19 diagnoses and an overall diagnostic yield of 54%. Diagnostic variants ranged from structural chromosomal abnormalities through to an intronic retrotransposon, disrupting splicing. Critical care management changed in 120 diagnosed patients (77%). This included major impacts, such as informing precision treatments, surgical and transplant decisions and palliation, in 94 patients (60%). Our results provide preliminary evidence of the clinical utility of integrating multi-omic approaches into mainstream diagnostic practice to fully realize the potential of rare disease genomic testing in a timely manner.

Grassland management crucially influences the delivery of ecosystem services from permanent grasslands. Variability in management practices is often described along a gradient from ‘low intensity’ to ‘high intensity’. These terms are likely to carry different meanings across European regions that differ inenvironmental and socio-economic conditions as well as between different groups of stakeholders. We conducted an online survey among grassland stakeholders asking them to characterise what they consider as ‘low’, ‘intermediate’ and ‘high’-intensity management in terms of cutting frequency, grazing intensity, and nitrogen fertilization. The answers of the 125 respondents revealed high variability in the thresholds between management intensity levels. Professional background (‘agriculture’ vs ‘ecology/ conservation’) explained only a small percentage of the variability. The biogeographical region on which the respondents’ expertise was based also influenced the evaluation of management practices. Our survey exposed the hidden problem of communicating about grassland management across regions and professional backgrounds, and identifies a need for a common terminology when making generalrecommendations for sustainable grassland management.

Objective: To study the epilepsy syndromes among the severe epilepsies of infancy and assess their incidence, etiologies, and outcomes. Method(s): A population-based cohort study was undertaken of severe epilepsies with onset before age 18 months in Victoria, Australia. Two epileptologists reviewed clinical features, seizure videos, and electroencephalograms to diagnose International League Against Epilepsy epilepsy syndromes. Incidence, etiologies, and outcomes at age 2 years were determined. Result(s): Seventy-three of 114 (64%) infants fulfilled diagnostic criteria for epilepsy syndromes at presentation, and 16 (14%) had "variants" of epilepsy syndromes in which there was one missing or different feature, or where all classical features had not yet emerged. West syndrome (WS) and "WS-like" epilepsy (infantile spasms without hypsarrhythmia or modified hypsarrhythmia) were the most common syndromes, with a combined incidence of 32.7/100 000 live births/year. The incidence of epilepsy of infancy with migrating focal seizures (EIMFS) was 4.5/100 000 and of early infantile epileptic encephalopathy (EIEE) was 3.6/100 000. Structural etiologies were common in "WS-like" epilepsy (100%), unifocal epilepsy (83%), and WS (39%), whereas single gene disorders predominated in EIMFS, EIEE, and Dravet syndrome. Eighteen (16%) infants died before age 2 years. Development was delayed or borderline in 85 of 96 (89%) survivors, being severe-profound in 40 of 96 (42%). All infants with EIEE or EIMFS had severe-profound delay or were deceased, but only 19 of 64 (30%) infants with WS, "WS-like," or "unifocal epilepsy" had severe-profound delay, and only two of 64 (3%) were deceased. Significance: Three quarters of severe epilepsies of infancy could be assigned an epilepsy syndrome or "variant syndrome" at presentation. In this era of genomic testing and advanced brain imaging, diagnosing epilepsy syndromes at presentation remains clinically useful for guiding etiologic investigation, i

BACKGROUND: GATA-binding protein 4 (GATA4) and Friend of GATA 2 protein (FOG2, also known as ZFPM2) form a heterodimer complex that has been shown to influence transcription of genes in a number of developmental systems. Recent evidence has also shown these genes play a role in gonadal sexual differentiation in humans. Previously we identified four variants in GATA4 and an unexpectedly large number of variants in ZFPM2 in a cohort of individuals with 46,XY Differences/Disorders of Sex Development (DSD) (Eggers et al, Genome Biology, 2016; 17: 243). METHOD(S): Here, we review variant curation and test the functional activity of GATA4 and ZFPM2 variants. We assess variant transcriptional activity on gonadal specific promoters (Sox9 and AMH) and variant protein-protein interactions. RESULT(S): Our findings support that the majority of GATA4 and ZFPM2 variants we identified are benign in their contribution to 46,XY DSD. Indeed, only one variant, in the conserved N-terminal zinc finger of GATA4, was considered pathogenic, with functional analysis confirming differences in its ability to regulate Sox9 and AMH and in protein interaction with ZFPM2. CONCLUSION(S): Our study helps define the genetic factors contributing to 46,XY DSD and suggests that the majority of variants we identified in GATA4 and ZFPM2/FOG2 are not causative.Copyright © 2019 The Murdoch Children's Research Institute. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

OBJECTIVE: To study the epilepsy syndromes among the severe epilepsies of infancy and assess their incidence, etiologies, and outcomes. METHODS: A population-based cohort study was undertaken of severe epilepsies with onset before age 18 months in Victoria, Australia. Two epileptologists reviewed clinical features, seizure videos, and electroencephalograms to diagnose International League Against Epilepsy epilepsy syndromes. Incidence, etiologies, and outcomes at age 2 years were determined. RESULTS: Seventy-three of 114 (64%) infants fulfilled diagnostic criteria for epilepsy syndromes at presentation, and 16 (14%) had "variants" of epilepsy syndromes in which there was one missing or different feature, or where all classical features had not yet emerged. West syndrome (WS) and "WS-like" epilepsy (infantile spasms without hypsarrhythmia or modified hypsarrhythmia) were the most common syndromes, with a combined incidence of 32.7/100 000 live births/year. The incidence of epilepsy of infancy with migrating focal seizures (EIMFS) was 4.5/100 000 and of early infantile epileptic encephalopathy (EIEE) was 3.6/100 000. Structural etiologies were common in "WS-like" epilepsy (100%), unifocal epilepsy (83%), and WS (39%), whereas single gene disorders predominated in EIMFS, EIEE, and Dravet syndrome. Eighteen (16%) infants died before age 2 years. Development was delayed or borderline in 85 of 96 (89%) survivors, being severe-profound in 40 of 96 (42%). All infants with EIEE or EIMFS had severe-profound delay or were deceased, but only 19 of 64 (30%) infants with WS, "WS-like," or "unifocal epilepsy" had severe-profound delay, and only two of 64 (3%) were deceased. SIGNIFICANCE: Three quarters of severe epilepsies of infancy could be assigned an epilepsy syndrome or "variant syndrome" at presentation. In this era of genomic testing and advanced brain imaging, diagnosing epilepsy syndromes at presentation remains clinically useful for guiding etiologic investigation, initi

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.

Introduction: Standard rapid genomic testing techniques analyse nuclear DNA variants using exome and/or genome sequencing (ES/GS). Rapid mtDNA analysis is not routinely available, particularly in centres performing ES, which does not deliver clinical-grade mtDNA sequencing. We describe our experience using rapid mtDNA sequencing in tandem with an ES-based rapid genomic diagnosis program as part of the Australian Genomics Acute Care flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid ES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): ES was non-diagnostic in both infants. mtDNA sequencing identified a single large mtDNA deletion in both infants, diagnostic of Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 hours 55 minutes and 54 hours 25 minutes, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with ES results in additional diagnoses in seriously ill children with suspected mitochondrial pathology, suggesting that ES alone may be insufficient in this setting. When designing rapid genomic diagnosis programs, centres should consider incorporating mtDNA amplification and analysis in individuals with suspected mitochondrial pathology, by combining ES and mtDNA sequencing in tandem, or analysing mtDNA data from GS, which captures the mitochondrial genome.

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

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

The Southern Ocean is characterized by longitudinal water circulations crossed by strong latitudinal gradients. How this oceanographic background shapes planktonic populations is largely unknown, despite the significance of this region for global biogeochemical cycles. Here, we show, based on genomic, morphometric, ecophysiological and mating compatibility data, an example of ecotypic differentiation and speciation within an endemic pelagic inhabitant, the diatom Fragilariopsis kerguelensis. We discovered three genotypic variants, one present throughout the latitudinal transect sampled, the others restricted to the north and south, respectively. The latter two showed reciprocal monophyly across all three genomes and significant ecophysiological differences consistent with local adaptation, but produced viable offspring in laboratory crosses. The third group was also reproductively isolated from the latter two. We hypothesize that this pattern originated by an adaptive expansion accompanied by ecotypic divergence, followed by sympatric speciation.

High concentrations of microplastics have been found in sea ice but the mechanisms by which they get captured into the ice and which role ice algae might play in this process remain unknown. Similarly, we do not know how the presence of microplastics might impact the colonization of sea ice by ice algae. To estimate the ecological impact of microplastics for Polar ecosystems, it is essential to understand their behaviour during ice formation and possible interactions with organisms inhabiting sea ice. In this study we tested the interaction between the ice algae Fragillariopsis cylindrus and microplastic beads with and without sea ice present and, in a third experiment, during the process of ice formation. With sea ice present, we found significantly less algae cells in the ice when incubated together with microplastics compared to the incubation without microplastics. However, during ice formation, the presence of microplastics did not impact the colonisation of the ice by F. cylindrus cells. Further, we observed a strong correlation between salinity and the relative amount of beads in the water and ice. With increasing salinity of the water, the relative amount of beads in the water decreased significantly. At the same time, the relative amount of beads in the ice increased significantly with increasing ice salinity. Both processes were not influenced by the presence of F. cylindrus. Also, we found indications that the presence of algae can affect the amount of microplastic beads sticking to the container walls. This could indicate that EPS produced by ice algae plays a significant role in surface binding properties of microplastics. Overall, our results highlight that the interactions between algae and microplastics have an influence on the uptake of microplastics into sea ice with possible implications for the sea ice food web.

BACKGROUND: GATA-binding protein 4 (GATA4) and Friend of GATA 2 protein (FOG2, also known as ZFPM2) form a heterodimer complex that has been shown to influence transcription of genes in a number of developmental systems. Recent evidence has also shown these genes play a role in gonadal sexual differentiation in humans. Previously we identified four variants in GATA4 and an unexpectedly large number of variants in ZFPM2 in a cohort of individuals with 46,XY Differences/Disorders of Sex Development (DSD) (Eggers et al, Genome Biology, 2016; 17: 243). METHOD: Here, we review variant curation and test the functional activity of GATA4 and ZFPM2 variants. We assess variant transcriptional activity on gonadal specific promoters (Sox9 and AMH) and variant protein-protein interactions. RESULTS: Our findings support that the majority of GATA4 and ZFPM2 variants we identified are benign in their contribution to 46,XY DSD. Indeed, only one variant, in the conserved N-terminal zinc finger of GATA4, was considered pathogenic, with functional analysis confirming differences in its ability to regulate Sox9 and AMH and in protein interaction with ZFPM2. CONCLUSIONS: Our study helps define the genetic factors contributing to 46,XY DSD and suggests that the majority of variants we identified in GATA4 and ZFPM2/FOG2 are not causative.

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.

Objective: While standard rapid genomic testing techniques analyse nuclear DNA variants using whole exome (WES) and/or whole genome (WGS) sequencing, rapid mtDNA analysis is not usually performed. We describe our experience using rapid mtDNA sequencing in tandem with WES in the Australian Genomics Acute Care Genomics flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid WES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): WES was non-diagnostic. mtDNA sequencing identified a single large-scale mtDNA deletion in both infants, consistent with Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 hours 55 minutes and 54 hours 25 minutes, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with WES Results in additional diagnoses in seriously ill children with suspected mitochondrial disease, suggesting that WES alone may be insufficient in this setting. When designing rapid genomic diagnostic programs, centres should consider mtDNA analysis, combining WES and mtDNA sequencing in tandem, or analysing mtDNA data from WGS, which captures the mitochondrial genome.Copyright © 2018

Introduction: Standard rapid genomic testing techniques analyze nuclear DNA variants using exome and/or genome sequencing (ES/GS). Rapid mtDNA analysis is not routinely available, particularly in centres performing ES, which does not deliver clinical-grade mtDNA sequencing. We describe our experience using rapid mtDNA sequencing in tandem with an ES-based rapid genomic diagnosis program as part of the Australian Genomics Acute Care flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid ES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): ES was non-diagnostic in both infants. mtDNA sequencing identified a single large mtDNA deletion in both infants, diagnostic of Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 h 55 min and 54 h 25 min, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with ES results in additional diagnoses in seriously ill children with suspected mitochondrial pathology, suggesting that ES alone may be insufficient in this setting. When designing rapid genomic diagnosis programs, centres should consider incorporating mtDNA amplification and analysis in individuals with suspected mitochondrial pathology, by combining ES and mtDNA sequencing in tandem, or analysing mtDNA data from GS, which captures the mitochondrial genome.

Rapid genomic testing is a valuable new diagnostic tool for acutely unwell infants, however exome sequencing does not deliver clinical-grade mitochondrial genome sequencing and may fail to diagnose mitochondrial disorders caused by mitochondrial DNA (mtDNA) variants. Rapid mitochondrial genome sequencing and analysis are not routinely available in rapid genomic diagnosis programmes. We present two critically ill neonates with transfusion-dependent anaemia and persistent lactic acidosis who underwent rapid mitochondrial genome sequencing in tandem with exome sequencing as part of an exome sequencing-based rapid genomic diagnosis programme. No diagnostic variants were identified on examination of the nuclear exome data for either infant. Mitochondrial genome sequencing identified a large mtDNA deletion in both infants, diagnosing Pearson syndrome within 74 and 55 h, respectively. Early diagnosis in the third week of life allowed the avoidance of a range of other investigations and appropriate treatment planning. Rapid mitochondrial genome analysis provides additional diagnostic and clinical utility and should be considered as an adjunct to exome sequencing in rapid genomic diagnosis programmes.Copyright © 2019, The Author(s), under exclusive licence to European Society of Human Genetics.

Fragilariopsis kerguelensis (O’ Meara) Hust. is a ubiquitous diatom of the Southern Ocean. Its thick frustules are the numerically dominant component of the siliceous sediment layer covering large parts of the seafloor beneath. Morphometric variability of frustules of this diatom has been of interest for paleoenvironmental reconstructions. Recently, two morphotypes differentiated by the morphometric descriptor rectangularity were described from a Southern Ocean sediment core, the relative abundance of which correlated with reconstructed paleotemperatures. In the present study, we use semi-automated microscopic and image analysis methods to answer whether these morphotypes also appear in recent assemblages, and if yes, do their distributions reflect geographic location or environmental factors. Three transects from the water column, sampled along the Greenwich meridian with hand nets, and one sediment surface transect from the South Pacific, were analyzed. In each of these transects, both morphotypes were detected, and annual mean sea surface temperatures (SST) were found to be a good predictor of their relative abundances. The transition between dominance of one or the other morphotype appeared roughly between the Antarctic Polar Front and the Southern Boundary of the Antarctic Circumpolar Current. Although more extensive circumpolar sampling will be needed to confirm the generality of our conclusions, the observed morphometric cline is a novel aspect of the biology of this species and can in the future potentially be used for further developing paleoproxies especially for highly F. kerguelensis-dominated sediment in the Southern Ocean.

BACKGROUND: Streptococcus pneumoniae is a significant global pathogen that colonises the nasopharynx of healthy children. Pneumococcal conjugate vaccines, which reduce nasopharyngeal colonisation of vaccine-type S. pneumoniae, may have broader effects on the nasopharyngeal microbiota; however, data are limited. In Fiji, nasopharyngeal carriage prevalence of S. pneumoniae and other colonising species differ between the two main ethnic groups. Here, we examined the association between the 7-valent pneumococcal conjugate vaccine (PCV7) and the nasopharyngeal microbiota of children in Fiji, including for each of the two main ethnic groups-indigenous Fijians (iTaukei) and Fijians of Indian descent (FID). METHOD: The nasopharyngeal microbiota of 132 Fijian children was examined using nasopharyngeal swabs collected from 12-month-old iTaukei and FID children who were vaccinated (3 doses PCV7) or unvaccinated in infancy as part of a phase II randomised controlled trial. Microbiota composition was determined by sequencing the V4 region of the 16S rRNA gene. Species-specific carriage of S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus was determined using real-time quantitative PCR. Associations between microbiota composition and other host and environmental factors were considered in the analysis. RESULTS: PCV7 had no overall impact on microbial diversity or composition. However, ethnic differences were observed in both diversity and composition with iTaukei children having higher relative abundance of Moraxella (p = 0.004) and Haemophilus (p = 0.004) and lower relative abundance of Staphylococcus (p = 0.026), Dolosigranulum (p = 0.004) and Corynebacterium (p = 0.003) compared with FID children. Further, when we stratified by ethnicity, associations with PCV7 could be detected: vaccinated iTaukei children had a lower relative abundance of Streptococcus and Haemophilus compared with unvaccinated iTaukei children (p = 0.022 and p = 0.043, r

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the DNA-binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti-testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females.

Using whole exome sequencing, we found a pathogenic variant in the EEF1A2 gene in a patient with a Rett syndrome-like (RTT-like) phenotype, further confirming the association between EEF1A2 and Rett syndrome RTT and RTT-like phenotypes.

The differential diagnostics in Rett syndrome has evolved with the development of next generation sequencing-based techniques and many patients have been diagnosed with other syndromes or variants in newly described genes where the associated phenotype(s) is yet to be fully explored. The term Rett-like refers to phenotypes with distinct overlapping features of Rett syndrome where the clinical criteria are not completely fulfilled. In this study we have combined a review of Rett-like disorders with data from a Danish cohort of 35 patients with Rett-like phenotypes emphasizing the diagnostic overlap with Pitt-Hopkins syndrome, Cornelia de Lange syndrome with SMC1A variants, and epileptic encephalopathies, for example, due to STXBP1 variants. We also found a patient with a pathogenic variant in KCNB1, which has not been previously linked to a Rett-like phenotype. This study underlines the clinical and genetic heterogeneity of a Rett syndrome spectrum, and provides an overview of the Rett syndrome-related genes described to date, and hence serves as a guide for diagnosing patients with Rett-like phenotypes.

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the

Variants in the NR5A1 gene encoding SF1 have been described in a diverse spectrum of disorders of sex development (DSD). Recently, we reported the use of a targeted gene panel for DSD where we identified 15 individuals with a variant in NR5A1, nine of which are novel. Here, we examine the functional effect of these changes in relation to the patient phenotype. All novel variants tested had reduced trans-activational activity, while several had altered protein level, localization, or conformation. In addition, we found evidence of new roles for SF1 protein domains including a region within the ligand binding domain that appears to contribute to SF1 regulation of Müllerian development. There was little correlation between the severity of the phenotype and the nature of the NR5A1 variant. We report two familial cases of NR5A1 deficiency with evidence of variable expressivity; we also report on individuals with oligogenic inheritance. Finally, we found that the nature of the NR5A1 variant does not inform patient outcomes (including pubertal androgenization and malignancy risk). This study adds nine novel pathogenic NR5A1 variants to the pool of diagnostic variants. It highlights a greater need for understanding the complexity of SF1 function and the additional factors that contribute.

OBJECTIVE: The severe epilepsies of infancy (SEI) are a devastating group of disorders that pose a major care and economic burden on society; early diagnosis is critical for optimal management. This study sought to determine the incidence and etiologies of SEI, and model the yield and cost-effectiveness of early genetic testing. METHODS: A population-based study was undertaken of the incidence, etiologies, and cost-effectiveness of a whole exome sequencing-based gene panel (targeted WES) in infants with SEI born during 2011-2013, identified through electroencephalography (EEG) and neonatal databases. SEI was defined as seizure onset before age 18 months, frequent seizures, epileptiform EEG, and failure of ≥2 antiepileptic drugs. Medical records, investigations, MRIs, and EEGs were analyzed, and genetic testing was performed if no etiology was identified. Economic modeling was performed to determine yield and cost-effectiveness of investigation of infants with unknown etiology at epilepsy onset, incorporating targeted WES at different stages of the diagnostic pathway. RESULTS: Of 114 infants with SEI (incidence = 54/100 000 live births/y), the etiology was determined in 76 (67%): acquired brain injuries (n = 14), focal cortical dysplasias (n = 14), other brain malformations (n = 17), channelopathies (n = 11), chromosomal (n = 9), metabolic (n = 6), and other genetic (n = 5) disorders. Modeling showed that incorporating targeted WES increased diagnostic yield compared to investigation without targeted WES (48/86 vs 39/86). Early targeted WES had lower total cost ($677 081 U.S. dollars [USD] vs $738 136 USD) than late targeted WES. A pathway with early targeted WES and limited metabolic testing yielded 7 additional diagnoses compared to investigation without targeted WES (46/86 vs 39/86), with lower total cost ($455 597 USD vs $661 103 USD), lower cost per diagnosis ($9904 USD vs $16 951 USD), and a dominant cost-effectiveness ratio. SIGNIFICANCE: Severe epilepsies occ

Remarkable progress has been achieved in understanding the mechanisms controlling sex determination, yet the cause for many Disorders of Sex Development (DSD) remains unknown. Of particular interest is a rare XX DSD subtype in which individuals are negative for SRY, the testis determining factor on the Y chromosome, yet develop testes or ovotestes, and both of these phenotypes occur in the same family. This is a naturally occurring disorder in humans (Homo sapiens) and dogs (C. familiaris). Phenotypes in the canine XX DSD model are strikingly similar to those of the human XX DSD subtype. The purposes of this study were to identify 1) a variant associated with XX DSD in the canine model and 2) gene expression alterations in canine embryonic gonads that could be informative to causation. Using a genome wide association study (GWAS) and whole genome sequencing (WGS), we identified a variant on C. familiaris autosome 9 (CFA9) that is associated with XX DSD in the canine model and in affected purebred dogs. This is the first marker identified for inherited canine XX DSD. It lies upstream of SOX9 within the canine ortholog for the human disorder, which resides on 17q24. Inheritance of this variant indicates that XX DSD is a complex trait in which breed genetic background affects penetrance. Furthermore, the homozygous variant genotype is associated with embryonic lethality in at least one breed. Our analysis of gene expression studies (RNA-seq and PRO-seq) in embryonic gonads at risk of XX DSD from the canine model identified significant RSPO1 downregulation in comparison to XX controls, without significant upregulation of SOX9 or other known testis pathway genes. Based on these data, a novel mechanism is proposed in which molecular lesions acting upstream of RSPO1 induce epigenomic gonadal mosaicism.