Genomic Diagnosis of Rare Pediatric Disease in the United Kingdom and Ireland.

BACKGROUND: Pediatric disorders include a range of highly penetrant, genetically heterogeneous conditions amenable to genomewide diagnostic approaches. Finding a molecular diagnosis is challenging but can have profound lifelong benefits. METHODS: We conducted a large-scale sequencing study involving more than 13,500 families with probands with severe, probably monogenic, difficult-to-diagnose developmental disorders from 24 regional genetics services in the United Kingdom and Ireland. Standardized phenotypic data were collected, and exome sequencing and microarray analyses were performed to investigate novel genetic causes. We developed an iterative variant analysis pipeline and reported candidate variants to clinical teams for validation and diagnostic interpretation to inform communication with families. Multiple regression analyses were performed to evaluate factors affecting the probability of diagnosis. RESULTS: A total of 13,449 probands were included in the analyses. On average, we reported 1.0 candidate variant per parent-offspring trio and 2.5 variants per singleton proband. Using clinical and computational approaches to variant classification, we made a diagnosis in approximately 41% of probands (5502 of 13,449). Of 3599 probands in trios who received a diagnosis by clinical assertion, approximately 76% had a pathogenic de novo variant. Another 22% of probands (2997 of 13,449) had variants of uncertain significance in genes that were strongly linked to monogenic developmental disorders. Recruitment in a parent-offspring trio had the largest effect on the probability of diagnosis (odds ratio, 4.70; 95% confidence interval [CI], 4.16 to 5.31). Probands were less likely to receive a diagnosis if they were born extremely prematurely (i.e., 22 to 27 weeks' gestation; odds ratio, 0.39; 95% CI, 0.22 to 0.68), had in utero exposure to antiepileptic medications (odds ratio, 0.44; 95% CI, 0.29 to 0.67), had mothers with diabetes (odds ratio, 0.52; 95% CI, 0.41 to 0.67), or were of African ancestry (odds ratio, 0.51; 95% CI, 0.31 to 0.78). CONCLUSIONS: Among probands with severe, probably monogenic, difficult-to-diagnose developmental disorders, multimodal analysis of genomewide data had good diagnostic power, even after previous attempts at diagnosis. (Funded by the Health Innovation Challenge Fund and Wellcome Sanger Institute.).

Pervasive lesion segregation shapes cancer genome evolution.

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

Transcription factor Wilms' tumor 1 regulates developmental RNAs through 3' UTR interaction.

Wilms' tumor 1 (WT1) is essential for the development and homeostasis of multiple mesodermal tissues. Despite evidence for post-transcriptional roles, no endogenous WT1 target RNAs exist. Using RNA immunoprecipitation and UV cross-linking, we show that WT1 binds preferentially to 3' untranslated regions (UTRs) of developmental targets. These target mRNAs are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Wt1 deletion leads to rapid turnover of specific mRNAs. WT1 regulates reporter gene expression through interaction with 3' UTR-binding sites. Combining experimental and computational analyses, we propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.

A dual role for the RNA helicase DHX34 in NMD and pre-mRNA splicing and its function in hematopoietic differentiation.

The DExD/H-box RNA helicase DHX34 is a nonsense-mediated decay (NMD) factor that together with core NMD factors coregulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using cross-linking immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon-intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial myelodysplasia (MDS)/acute myeloid leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggest that alterations in the levels and/or activity of DHX34 could contribute to human disease.

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands.

Making new genetic diagnoses with old data: iterative reanalysis and reporting from genome-wide data in 1,133 families with developmental disorders.

PURPOSE: Given the rapid pace of discovery in rare disease genomics, it is likely that improvements in diagnostic yield can be made by systematically reanalyzing previously generated genomic sequence data in light of new knowledge. METHODS: We tested this hypothesis in the United Kingdom-wide Deciphering Developmental Disorders study, where in 2014 we reported a diagnostic yield of 27% through whole-exome sequencing of 1,133 children with severe developmental disorders and their parents. We reanalyzed existing data using improved variant calling methodologies, novel variant detection algorithms, updated variant annotation, evidence-based filtering strategies, and newly discovered disease-associated genes. RESULTS: We are now able to diagnose an additional 182 individuals, taking our overall diagnostic yield to 454/1,133 (40%), and another 43 (4%) have a finding of uncertain clinical significance. The majority of these new diagnoses are due to novel developmental disorder-associated genes discovered since our original publication. CONCLUSION: This study highlights the importance of coupling large-scale research with clinical practice, and of discussing the possibility of iterative reanalysis and recontact with patients and health professionals at an early stage. We estimate that implementing parent-offspring whole-exome sequencing as a first-line diagnostic test for developmental disorders would diagnose >50% of patients.

The risk of non-union per fracture: current myths and revised figures from a population of over 4 million adults.

Background and purpose - Fracture non-union remains a major clinical problem, yet there are no data available regarding the overall risk of fractures progressing to non-union in a large population. We investigated the rate of non-union per fracture in a large adult population. Methods - National data collected prospectively over a 5-year period and involving just under 5,000 non-unions were analyzed and compared to the incidence of fracture in the same period. Results and interpretation - The overall risk of non-union per fracture was 1.9%, which is considerably less than previously believed. However, for certain fractures in specific age groups the risk of non-union rose to 9%. As expected, these higher rates of non-union were observed with tibial and clavicular fractures, but-less expectedly-it was in the young and middle-aged adults rather than in the older and elderly population. This study is the first to examine fracture non-union rates in a large population according to age and site, and provides more robust (and lower) estimates of non-union risk than those that are frequently quoted.

An evaluation of the Herscovici classification for fractures of the medial malleolus.

BACKGROUND: Despite its use in the literature, the application of the Herscovici classification system for medial malleolus fractures has not been evaluated. METHODS: We aimed to determine the reliability and accuracy of the Herscovici classification. The blinded radiographs of 130 patients were independently classified by four orthopaedic trauma surgeons. We held a consensus meeting where observers agreed on a final classification and this served as our reference standard. We used weighted kappa (κ) coefficients of agreement. RESULTS: Twenty-four fractures (18%) were deemed unclassifiable. The classification system demonstrated moderate inter-observer reliability (κ=0.54, 95% CI 0.40-0.68) but substantial reproducibility (κ=0.64, 95% CI 0.51-0.79). Accuracy, when compared with the reference standard, was κ=0.54 (95% CI 0.40-0.66). CONCLUSIONS: The obliquity of the fracture line, and fracture extension, created difficulty in classification in 26% of cases. 18% of our cases could not be classified by majority decision. Our results emphasise the challenges faced in classifying these fractures. Future work should focus on refining the Herscovici classification.

Medium-term patient-reported outcomes after total hip replacement for displaced hip fractures.

INTRODUCTION: The aim of the present study was to define the medium-term outcomes following total hip replacement (THR) for hip fracture. METHODS: We prospectively followed up 92 patients who underwent THR for a displaced hip fracture over a 3-year period between 2007 and 2010. These patients were followed up at 5 years using the Oxford Hip Score, Short-Form 12 (SF-12) questionnaire and satisfaction questionnaire. These outcomes were compared to the short-term outcomes previously reported at 2 years to determine any significant differences. RESULTS: Mean follow-up was at 5.4 years with a mean age at follow-up of 76.5 years. Seventy-four patients (80%) responded. Patients reported excellent functional outcomes and satisfaction (mean Oxford Hip Score 40.3; SF-12 Physical Health Composite Score 44.0; SF-12 Mental Health Composite Score 46.2; mean satisfaction 90%). The rates of dislocation (2%), deep infection (2%) and revision (3%) were comparable to those quoted for elective THR. When compared with 2-year follow-up, there were no statistically significant adverse changes in outcome parameters. CONCLUSIONS: Medium-term outcomes for THR after hip fracture in fit older patients are excellent, and these results demonstrate that the early proven benefits of this surgery are sustained into the midterm.

Dizzy-Beats: a Bayesian evidence analysis tool for systems biology.

MOTIVATION: Model selection and parameter inference are complex problems of long-standing interest in systems biology. Selecting between competing models arises commonly as underlying biochemical mechanisms are often not fully known, hence alternative models must be considered. Parameter inference yields important information on the extent to which the data and the model constrain parameter values. RESULTS: We report Dizzy-Beats, a graphical Java Bayesian evidence analysis tool implementing nested sampling - an algorithm yielding an estimate of the log of the Bayesian evidence Z and the moments of model parameters, thus addressing two outstanding challenges in systems modelling. A likelihood function based on the L1-norm is adopted as it is generically applicable to replicated time series data. AVAILABILITY AND IMPLEMENTATION: http://sourceforge.net/p/bayesevidence/home/Home/.

Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation.

Mammalian chromosomes fold into arrays of megabase-sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher-order organization remains elusive. Here, we investigate TAD higher-order interactions with Hi-C through neuronal differentiation and show that they form a hierarchy of domains-within-domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree-like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency.

Transcriptional dynamics reveal critical roles for non-coding RNAs in the immediate-early response.

The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE) time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs) and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset.

Patient satisfaction from two studies of collaborative doctor-pharmacist prescribing in Australia.

BACKGROUND: Pharmacist prescribing has been introduced in several countries and is a possible future role for pharmacy in Australia. OBJECTIVE: To assess whether patient satisfaction with the pharmacist as a prescriber, and patient experiences in two settings of collaborative doctor-pharmacist prescribing may be barriers to implementation of pharmacist prescribing. DESIGN: Surveys containing closed questions, and Likert scale responses, were completed in both settings to investigate patient satisfaction after each consultation. A further survey investigating attitudes towards pharmacist prescribing, after multiple consultations, was completed in the sexual health clinic. SETTING AND PARTICIPANTS: A surgical pre-admission clinic (PAC) in a tertiary hospital and an outpatient sexual health clinic at a university hospital. Two hundred patients scheduled for elective surgery, and 17 patients diagnosed with HIV infection, respectively, recruited to the pharmacist prescribing arm of two collaborative doctor-pharmacist prescribing studies. RESULTS: Consultation satisfaction response rates in PAC and the sexual health clinic were 182/200 (91%) and 29/34 (85%), respectively. In the sexual health clinic, the attitudes towards pharmacist prescribing survey response rate were 14/17 (82%). Consultation satisfaction was high in both studies, most patients (98% and 97%, respectively) agreed they were satisfied with the consultation. In the sexual health clinic, all patients (14/14) agreed that they trusted the pharmacist's ability to prescribe, care was as good as usual care, and they would recommend seeing a pharmacist prescriber to friends. DISCUSSION AND CONCLUSION: Most of the patients had a high satisfaction with pharmacist prescriber consultations, and a positive outlook on the collaborative model of care in the sexual health clinic.

Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution.

Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high-throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3' major domain of the 20S pre-ribosomal RNA.

Stochastic EM-based TFBS motif discovery with MITSU.

MOTIVATION: The Expectation-Maximization (EM) algorithm has been successfully applied to the problem of transcription factor binding site (TFBS) motif discovery and underlies the most widely used motif discovery algorithms. In the wider field of probabilistic modelling, the stochastic EM (sEM) algorithm has been used to overcome some of the limitations of the EM algorithm; however, the application of sEM to motif discovery has not been fully explored. RESULTS: We present MITSU (Motif discovery by ITerative Sampling and Updating), a novel algorithm for motif discovery, which combines sEM with an improved approximation to the likelihood function, which is unconstrained with regard to the distribution of motif occurrences within the input dataset. The algorithm is evaluated quantitatively on realistic synthetic data and several collections of characterized prokaryotic TFBS motifs and shown to outperform EM and an alternative sEM-based algorithm, particularly in terms of site-level positive predictive value. AVAILABILITY AND IMPLEMENTATION: Java executable available for download at http://www.sourceforge.net/p/mitsu-motif/, supported on Linux/OS X.

The importance of the orthopaedic doctors' appearance: a cross-regional questionnaire based study.

OBJECTIVES: Critics of the Department of Health 'bare below the elbow' guidelines have raised concerns over the impact of these dress regulations on the portrayed image and professionalism of doctors. However, the importance of the doctor's appearance in relation to other professional attributes is largely unknown. The purpose of this study was to determine the opinion of patients on the importance of appearance and the style of clothing worn by doctors. DESIGN: Patient questionnaire survey, administered across four Scottish regions. SETTING: Orthopaedic outpatient departments. PARTICIPANTS: 427 patients and accompanying relatives. MAIN OUTCOME MEASURES: The absolute and relative importance of the doctors' appearance, as reported using a 5-point Likert scale. The absolute and relative importance of the style of clothing worn by doctors, as reported using a 5-point Likert scale. The rank preferences for four different styles of doctors' attire as illustrated by standardised clinical photographs. RESULTS: The study was appropriately powered to identify a 0.5 difference in mean rank values with 0.90 power at a = 0.05. The majority of participants felt the doctors' appearance was important but not as important as compassion, politeness and knowledge. Only 50% felt that the style of doctors clothing mattered; what proved more important was an impression of cleanliness and good personal hygiene. In terms of how patients would prefer doctors to dress in clinic, the most popular choice proved to be the smart casual style of dress, which conforms with the 'bare below the elbows' dress code policy. The smart casual clothing style was the highest ranked choice irrespective of patient age, gender, regional or socioeconomic background. CONCLUSIONS: The doctors' appearance is of importance to patients and their relatives, but they view many other attributes as more important than how we choose to dress. While not specifically addressing the role of doctors clothing in the transmission of infection, our results do support the preference of patients for 'bare below the elbows' workplace attire.

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Fostering Gender-IQ: Barriers and Enablers to Gender-affirming Behavior Amongst an Australian General Practitioner Cohort.

While the visible population of trans and gender diverse Australians has grown significantly in recent years, primary health-care access remains hindered by a lack of practitioner competency and stigmatization. This article draws on qualitative research of purposively selected gender-affirming general practitioners (GPs) in Australia to explore barriers, and enablers when treating trans and gender diverse patients. Perspectives and behaviors during the gender-affirming clinical encounter were theoretically informed through minority stress theory, and master narrative frameworks. Reflexive thematic analysis facilitated a rich description of exemplary gender-affirming primary care. A considerable gap exists between structural, clinical, and cultural behaviors among competent gender-affirming GPs in Australia, and the majority of practitioners evidenced in the literature. This critical analysis contributes to better understanding how gender-affirming Australian GPs diffuse minority stress, negotiate cis-normative biases, and foster a person-centered longitudinal therapeutic relationship with their trans and gender diverse patients. An encounter the article argues may also provide an essential buffer for GPs in Australia against the risk of professional burnout. Gender-affirming practice should be taught as a core competency and be required as professional development for GPs in Australia, to ensure a beneficial clinical encounter for the growing trans and gender diverse population.

IMPROVE-DD: Integrating multiple phenotype resources optimizes variant evaluation in genetically determined developmental disorders.

Diagnosing rare developmental disorders using genome-wide sequencing data commonly necessitates review of multiple plausible candidate variants, often using ontologies of categorical clinical terms. We show that Integrating Multiple Phenotype Resources Optimizes Variant Evaluation in Developmental Disorders (IMPROVE-DD) by incorporating additional classes of data commonly available to clinicians and recorded in health records. In doing so, we quantify the distinct contributions of sex, growth, and development in addition to Human Phenotype Ontology (HPO) terms and demonstrate added value from these readily available information sources. We use likelihood ratios for nominal and quantitative data and propose a classifier for HPO terms in this framework. This Bayesian framework results in more robust diagnoses. Using data systematically collected in the Deciphering Developmental Disorders study, we considered 77 genes with pathogenic/likely pathogenic variants in ≥10 individuals. All genes showed at least a satisfactory prediction by receiver operating characteristic when testing on training data (AUC ≥ 0.6), and HPO terms were the best predictor for the majority of genes, though a minority (13/77) of genes were better predicted by other phenotypic data types. Overall, classifiers based upon multiple integrated phenotypic data sources performed better than those based upon any individual source, and importantly, integrated models produced notably fewer false positives. Finally, we show that IMPROVE-DD models with good predictive performance on cross-validation can be constructed from relatively few individuals. This suggests new strategies for candidate gene prioritization and highlights the value of systematic clinical data collection to support diagnostic programs.