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Renal Fanconi syndrome (RFS) is a generalised disorder of the proximal convoluted tubule. Many genes have been associated with RFS including those that cause systemic disorders such as cystinosis, as well as isolated RFS. We discuss the case of a 10-year-old female who presented with leg pain and raised creatinine on a screening blood test. Her mother has RFS and required a kidney transplant in her thirties. Further investigations confirmed RFS in the daughter. Exome sequencing was performed on the affected mother, child, and unaffected father. We identified a novel variant in GATM; c.965GC p.(Arg322Pro) segregating dominantly in the mother and daughter. We validated our finding with molecular dynamics simulations and demonstrated a dynamic signature that differentiates our variant and two previously identified pathogenic variants in GATM from wildtype. Genetic testing has uncovered a novel pathogenic variant that predicts progression to end stage kidney failure and has important implications for family planning and cascade testing. We recommend that GATM is screened for in children presenting with RFS, in addition to adults, particularly with kidney failure, who may have had previous negative gene testing.

Background Crohn’s disease (CD) is highly heterogenous and may be complicated by stricturing behavior. Personalized prediction of stricturing will inform management. We aimed to create a stricturing risk stratification model using genomic/clinical data. Methods Exome sequencing was performed on CD patients, and phenotype data retrieved. Biallelic variants in NOD2 were identified. NOD2 was converted into a per-patient deleteriousness metric (“GenePy”). Using training data, patients were stratified into risk groups for fibrotic stricturing using NOD2. Findings were validated in a testing data set. Models were modified to include disease location at diagnosis. Cox proportional hazards assessed performance. Results Six hundred forty-five patients were included (373 children and 272 adults); 48 patients fulfilled criteria for monogenic NOD2-related disease (7.4%), 24 of whom had strictures. NOD2 GenePy scores stratified patients in training data into 2 risk groups. Within testing data, 30 of 161 patients (18.6%) were classified as high-risk based on the NOD2 biomarker, with stricturing in 17 of 30 (56.7%). In the low-risk group, 28 of 131 (21.4%) had stricturing behavior. Cox proportional hazards using the NOD2 risk groups demonstrated a hazard ratio (HR) of 2.092 (P = 2.4 × 10-5), between risk groups. Limiting analysis to patients diagnosed aged Conclusions A NOD2 genomic biomarker predicts stricturing risk, with prognostic power improved in pediatric-onset CD. Implementation into a clinical setting can help personalize management.

PURPOSE: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. METHOD: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). RESULTS: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had identifier with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. CONCLUSION: Pathogenic WNK3 variants cause a rare form of human X-linked identifier with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.

Studies of Crohn’s disease have consistently implicated NOD2 as the most important gene in disease pathogenesis since first being identified in 2001. Thereafter, genome-wide association, next-generation sequencing and functional analyses have all confirmed a key role for NOD2, but despite this, NOD2 also has significant unresolved complexity. More recent studies have reinvigorated an early hypothesis that NOD2 may be a single-gene cause of disease, and the distinct ileal stricturing phenotype seen with NOD2-related disease presents an opportunity for personalized diagnosis, disease prediction and targeted therapy. The genomics of NOD2 has much that remains unknown, including the role of rare variation, phasing of variants across the haplotype block and the role of variation in the NOD2-regulatory regions. Here, we discuss the evidence and the unmet needs of NOD2 research, based on recently published evidence, and suggest methods that may meet these requirements.

TATA-binding protein associated factor 4 (TAF4) is a subunit of the Transcription Factor IID (TFIID) complex, a central player in transcription initiation. Other members of this multimeric complex have been implicated previously as monogenic disease genes in human developmental disorders. TAF4 has not been described to date as a monogenic disease gene. We here present a cohort of eight individuals, each carrying de novo putative loss-of-function (pLoF) variants in TAF4 and expressing phenotypes consistent with a neuro-developmental disorder (NDD). Common features include intellectual disability, abnormal behavior, and facial dysmorphisms. We propose TAF4 as a novel dominant disease gene for NDD, and coin this novel disorder “TAF4-related NDD” (T4NDD). We place T4NDD in the context of other disorders related to TFIID subunits, revealing shared features of T4NDD with other TAF-opathies.

PurposeExome and genome sequencing have drastically accelerated novel disease gene discoveries. However, discovery is still hindered by myriad variants of uncertain significance found in genes of undetermined biological function. This necessitates intensive functional experiments on genes of equal predicted causality, leading to a major bottleneck.MethodsWe apply the loss-of-function observed/expected upper-bound fraction metric of intolerance to gene inactivation to curate a list of predicted haploinsufficient disease genes. Using data from the 100,000 Genomes Project, we adopt a gene-to-patient approach that matches de novo loss-of-function variants in constrained genes to patients with rare disease. Through large-scale aggregation of data, we reduce excess analytical noise currently hindering novel discoveries.ResultsResults from 13,949 trios revealed 643 rare, de novo predicted loss-of-function events filtered from 1044 loss-of-function observed/expected upper-bound fraction–constrained genes. A total of 168 variants occurred within 126 genes without a known disease-gene relationship. Of these, 27 genes had >1 kindred affected, and for 18 of these genes, multiple kindreds had overlapping phenotypes. Two years after initial analysis, 11 of 18 (61%) of these genes have been independently published as novel disease gene discoveries.ConclusionUsing large cohorts and adopting gene-based approaches can rapidly and objectively accelerate dominantly inherited novel gene discovery by targeting the most appropriate genes for functional validation.

Background/Objective– Heterogeneity and chronicity of Crohn’s disease (CD) make prediction of outcomes difficult. To date, no longitudinal measure can quantify burden over a patient’s disease course, preventing assessment and integration into predictive modelling. Here, we aimed to demonstrate the feasibility of constructing a data driven, longitudinal disease burden score.Methods– Literature was reviewed for tools used in assessment of CD activity. Themes were identified to construct a paediatric CD morbidity index (PCD-MI). Scores were assigned to variables. Data were extracted automatically from the electronic patient records at Southampton Children’s Hospital, diagnosed from 2012 to 2019 (inclusive). PCD-MI scores were calculated, adjusted for duration of follow up and assessed for variation (ANOVA) and distribution (Kolmogorov-Smirnov).Results– Nineteen clinical/biological features across five themes were included in the PCD-MI including blood/faecal/radiological/endoscopic results, medication usage, surgery, growth parameters and extraintestinal manifestations. Maximal score was 100 after accounting for follow-up duration.PCD-MI was assessed in 66 patients, mean age 12.5 years. Following quality filtering, 9528 blood/faecal test results and 1309 growth measures were included. Mean PCD-MI score was 14.95 (range 2.2-32.5), data were normally distributed (p=0.2) with 25% of patients having a PCD-MI Conclusions– PCD-MI is a calculatable measure for a cohort of patients diagnosed over an 8-year period, integrating a wide-range of data with potential to determine high or low disease burden. Future iterations of the PCD-MI require refinement of included features, optimised scores and validation on external cohorts.

Predicted loss of function (pLoF) variants are highly deleterious and play an important role in disease biology, but many of these variants may not actually result in loss-of-function. Here we present a framework that advances interpretation of pLoF variants in research and clinical settings by considering three categories of LoF evasion: (1) predicted rescue by secondary sequence properties, (2) uncertain biological relevance, and (3) potential technical artifacts. We also provide recommendations on adjustments to ACMG/AMP guidelines’s PVS1 criterion. Applying this framework to all high-confidence pLoF variants in 22 autosomal recessive disease-genes from the Genome Aggregation Database (gnomAD, v2.1.1) revealed predicted LoF evasion or potential artifacts in 27.3% (304/1,113) of variants. The major reasons were location in the last exon, in a homopolymer repeat, in low per-base expression (pext) score regions, or the presence of cryptic splice rescues. Variants predicted to be potential artifacts or to evade LoF were enriched for ClinVar benign variants. PVS1 was downgraded in 99.4% (162/163) of LoF evading variants assessed, with 17.2% (28/163) downgraded as a result of our framework, adding to previous guidelines. Variant pathogenicity was affected (mostly from likely pathogenic to VUS) in 20 (71.4%) of these 28 variants. This framework guides assessment of pLoF variants beyond standard annotation pipelines, and substantially reduces false positive rates, which is key to ensure accurate LoF variant prediction in both a research and clinical setting.

Genome sequencing is now available as a clinical test on the National Health Service (NHS) through the Genome Medicine Service (GMS). The GMS have set out an analytical strategy that predominantly filters genome data on a pre-selected gene panel(s). Whilst this approach reduces the number of variants requiring assessment by reporting laboratories, pathogenic variants outside of the gene panel applied may be missed, and candidate variants in novel genes are largely ignored.This study sought to compare a research exome analysis to an independent clinical genome analysis performed through the NHS for the same group of patients. When analysing the exome data, we applied a panel agnostic approach filtering for variants withHighPathogenicPotential (HiPPo) using ClinVar, allele frequency, andin silicoprediction tools. We then compared this gene agnostic analysis to the panel-based approach as applied by the GMS to genome data. Later we restricted HiPPo variants to a panel of the Gene Curation Coalition (GenCC) morbid genes and compared the diagnostic yield with the variants filtered using the GMS strategy.24 patients from 8 families underwent parallel research exome sequencing and GMS genome sequencing. HiPPo analysis applied to research exome data identified a similar number of variants as the gene panel-based approach applied by the GMS. GMS clinical genome analysis identified and returned 2 pathogenic variants and 3 variants of uncertain significance. HiPPo research exome analysis identified the same variants plus an additional pathogenic variant and a further 3de novovariants of uncertain significance in novel genes, where case series and functional studies are underway. When HiPPo was restricted to GenCC disease genes (strong or definitive), the same pathogenic variants were identified yet statistically fewer variants required assessment to identify more diagnostic variants than reported by the GMS genome strategy. This gave a diagnostic rate per variant assessed of 20% for HiPPo restricted to GenCC versus 3% for the GMS panel-based approach. With plans to sequence 5 million more NHS patients, strategies are needed to optimise the full potential of genome data beyond gene panels whilst minimising the burden of variants that require clinical assessment.

SRRM2 encodes a splicing factor recently implicated in developmental disorders due to a statistical enrichment of de novo mutations. Using data from the 100,000 Genomes Project, four unrelated individuals with intellectual disability (ID) were identified, each harbouring de novo whole gene deletions of SRRM2. Deletions ranged between 248 and 482 kb in size and all distal breakpoints clustered within a complex 144 kb palindrome situated 75 kb upstream of SRRM2. Strikingly, three of the deletions were complex, with inverted internal segments of 45-94 kb. In one proband-mother duo, de novo status was inferred by haplotype analysis. Together with two additional patients who harboured smaller predicted protein-truncating variants (p.Arg632 ∗ and p.Ala2223Leufs ∗ 13), we estimate the prevalence of this condition in cohorts of patients with unexplained ID to be ~1/1300. Phenotypic blending, present for two cases with additional pathogenic variants in CASR/PKD1 and SLC17A5, hampered the phenotypic delineation of this recently described condition. Our data highlights the benefits of genome sequencing for resolving structural complexity and inferring de novo status. The genomic architecture of 16p13.3 may give rise to relatively high rates of complex rearrangements, adding to the list of loci associated with recurrent genomic disorders.

BACKGROUND: The U.K. 100,000 Genomes Project is in the process of investigating the role of genome sequencing in patients with undiagnosed rare diseases after usual care and the alignment of this research with health care implementation in the U.K. National Health Service. Other parts of this project focus on patients with cancer and infection.METHODS: We conducted a pilot study involving 4660 participants from 2183 families, among whom 161 disorders covering a broad spectrum of rare diseases were present. We collected data on clinical features with the use of Human Phenotype Ontology terms, undertook genome sequencing, applied automated variant prioritization on the basis of applied virtual gene panels and phenotypes, and identified novel pathogenic variants through research analysis.RESULTS: Diagnostic yields varied among family structures and were highest in family trios (both parents and a proband) and families with larger pedigrees. Diagnostic yields were much higher for disorders likely to have a monogenic cause (35%) than for disorders likely to have a complex cause (11%). Diagnostic yields for intellectual disability, hearing disorders, and vision disorders ranged from 40 to 55%. We made genetic diagnoses in 25% of the probands. A total of 14% of the diagnoses were made by means of the combination of research and automated approaches, which was critical for cases in which we found etiologic noncoding, structural, and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohortwide burden testing across 57,000 genomes enabled the discovery of three new disease genes and 19 new associations. Of the genetic diagnoses that we made, 25% had immediate ramifications for clinical decision making for the patients or their relatives.CONCLUSIONS: Our pilot study of genome sequencing in a national health care system showed an increase in diagnostic yield across a range of rare diseases. (Funded by the National Institute for Health Research and others.).

Genome sequencing was first offered clinically in the UK through the 100,000 Genomes Project (100KGP). Analysis was restricted to predefined gene panels associated with the patient's phenotype. However, panels rely on clearly characterised phenotypes and risk missing diagnoses outside of the panel(s) applied. We propose a complementary method to rapidly identify pathogenic variants, including those missed by 100KGP methods.The Loss-of-function Observed/Expected Upper-bound Fraction (LOEUF) score quantifies gene constraint, with low scores correlated with haploinsufficiency. We applied DeNovoLOEUF, a filtering strategy to sequencing data from 13,949 rare disease trios in the 100KGP, by filtering for rare, de novo, loss-of-function variants in disease genes with a LOEUF score 0.2. We compared our findings with the corresponding patient's diagnostic reports.324/332 (98%) of the variants identified using DeNovoLOEUF were diagnostic or partially diagnostic (whereby the variant was responsible for some of the phenotype). We identified 39 diagnoses that were "missed" by 100KGP standard analyses, which are now being returned to patients.We have demonstrated a highly specific and rapid method with a 98% positive predictive value that has good concordance with standard analysis, low false-positive rate, and can identify additional diagnoses. Globally, as more patients are being offered genome sequencing, we anticipate that DeNovoLOEUF will rapidly identify new diagnoses and facilitate iterative analyses when new disease genes are discovered.

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10(−11)). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10(−15)). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.

Background Neurosurgery is a notoriously difficult career to enter and requires medical students to engage in extracurricular activities to demonstrate their commitment to the specialty. The National Undergraduate Neuroanatomy Competition (NUNC) was established in 2013 as a means for students to display this commitment as well as academic ability. Methods A bespoke 22-item questionnaire was designed to determine career outcomes and the role of competition attendance in job applications. It was distributed using the SurveyMonkey website to the 87 attendees at the 2013 and 2014 competitions. Results Responses were received by 40 competitors (response rate, 46.0%). Twenty-four responders (60.0%) intended to pursue a career in either neurosurgery (n = 18) or neurology (n = 6). This included 10 responders (25.0%) who had successfully entered either neurosurgery (n = 9) or neurology (n = 1). The performance of these 10 was significantly better than the other responders (57.0 ± 13.6% vs. 46.5 ± 13.5% [n = 30]; P = 0.036). Seventeen responders (42.5%) either included their attendance at NUNC in a post-Foundation job application or intend to. Conclusions The NUNC provides the opportunity for medical students to demonstrate their interest in neurosurgery. It has the potential to be used as a tool for recognizing medical students suitable for neurosurgery training.

Accurate regulation of cutaneous immunity is fundamental for human health and quality of life but is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance vs inflammation in human skin, we set up a human in vivo allergen challenge study, exposing patients with atopic dermatitis (AD) to house dust mite (HDM). Analyses of transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of resident and infiltrating immune cells indicated that inflammatory responses to HDM were associated with immune activation in Langerhans cells (LCs) and cutaneous T cells. High basal level of TNFα production by cutaneous Th17 T cells predisposed to an inflammatory reaction and resulted in formation of hub structures where LCs and T cells interacted, leading to loss of functional programming in LCs. Additionally, single nucleotide polymorphisms in MT1X gene associated with enhanced expression of metallothioneins and transcriptional programmes encoding antioxidant defences across skin cell types in non-reactive patients, were protective against T cell mediated inflammation. Our results provide a unique insight into the dynamics of immune regulation in the human skin and define regulatory circuits that can be harnessed to improve skin health and treat disease.

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes

Kawasaki disease (KD) is a paediatric vasculitis associated with coronary artery aneurysms (CAA). Genetic variants influencing susceptibility to KD have been previously identified, but no risk alleles have been validated that influence CAA formation. We conducted a genome-wide association study (GWAS) for CAA in KD patients of European descent with 200 cases and 276 controls. A second GWAS for susceptibility pooled KD cases with healthy paediatric controls from vaccine trials in the UK (n = 1609). Logistic regression mixed models were used for both GWASs. The susceptibility GWAS was meta-analysed with 400 KD cases and 6101 controls from a previous European GWAS, these results were further meta-analysed with Japanese GWASs at two putative loci. The CAA GWAS identified an intergenic region of chromosome 20q13 with multiple SNVs showing genome-wide significance. The risk allele of the most associated SNV (rs6017006) was present in 13% of cases and 4% of controls; in East Asian 1000 Genomes data, the allele was absent or rare. Susceptibility GWAS with meta-analysis with previously published European data identified two previously associated loci (ITPKC and FCGR2A). Further meta-analysis with Japanese GWAS summary data from the CASP3 and FAM167A genomic regions validated these loci in Europeans showing consistent effects of the top SNVs in both populations. We identified a novel locus for CAA in KD patients of European descent. The results suggest that different genes determine susceptibility to KD and development of CAA and future work should focus on the function of the intergenic region on chromosome 20q13.

Next generation sequencing (NGS) has revolutionised rare disease diagnostics. Concomitant with advancing technologies has been a rise in the number of new gene disorders discovered and diagnoses made for patients and their families. However, despite the trend towards whole exome and whole genome sequencing, diagnostic rates remain suboptimal. On average, only ~30% of patients receive a molecular diagnosis. National sequencing projects launched in the last 5 years are integrating clinical diagnostic testing with research avenues to widen the spectrum of known genetic disorders. Consequently, efforts to diagnose genetic disorders in a clinical setting are now often shared with efforts to prioritise candidate variants for the detection of new disease genes. Herein we discuss some of the biggest obstacles precluding molecular diagnosis and discovery of new gene disorders. We consider bioinformatic and analytical challenges faced when interpreting next generation sequencing data and showcase some of the newest tools available to mitigate these issues. We consider how incomplete penetrance, non-coding variation and structural variants are likely to impact diagnostic rates, and we further discuss methods for uplifting novel gene discovery by adopting a gene-to-patient-based approach.

Introduction: Aryl hydrocarbon nuclear translocator 2 (ARNT2) is a basic helix-loop-helix (bHLH)-PAS (Per/Arnt/Sim) transcription factor shown to be critical to the development of paraventricular nucleus of the hypothalamus (PVN), key region for energy homeostasis and feeding response. In vivo and in vitro studies have shown that ARNT2 is an obligate heterodimer for SIM1, known cause of monogenic obesity. Null mutations in Arnt2 in animals are not viable, but hypomorphic mutation results in hyperphagic obesity and its associated consequences (1). Due to the critical role of ARNT2 in the development of PVN, we hypothesize that hypomorphic mutations may result in early onset obesity in humans. Methods: The Genetics of Early Childhood Obesity (GECO) study recruits children with severe obesity (BMI > 120% of 95th percentile) of early onset (< 6 years). Whole exome sequencing (WES) was performed in a subset of proband-parent trios. The functional validation of the mutation(s) in ARNT2 is ongoing with co-transfection of tagged Arnt2 and Sim1 in HEK293 cells, with the induction of a luciferase reporter gene under the control of 6 repeats of bHLH-PAS core binding element by the Arnt2-Sim1 complex. Results: Two adolescents from unrelated families were found to have genetic variants in ARNT2. Subject 1 has a novel de novo heterozygous coding variant in ARNT2, c.388 C>G (p.P130A, CADD 25), predicted to be deleterious by 8/12 in silico algorithms. She is a 14-year old Caucasian girl with severe early onset obesity, BMI 28.1 kg/m2 (BMIz +4.72) at 2.5 years of age that has increased to 53.54 kg/m2 (BMIz + 3.25) at 14-years, and height > 95th %tile. She is non-dysmorphic, has developmental delay, absence seizures, behavior abnormalities & glucose intolerance/dyslipidemia secondary to obesity. Using genematcher, we identified another proband with the phenotype of obesity: an African American girl (BMIz +1.9) with biallelic inherited heterozygous variants in ARNT2, c.1228T>A (p.W410R, CADD 29) and c.916G>A (p.G306S, CADD 22). An only child conceived by IVF, she is non-dysmorphic and on treatment for bilateral focal epilepsy. All 3 variants are rare, with mean allele frequency < 0.005 in population-based databases such as gNOMAD. Both the patients have early onset obesity and a significant neurological phenotype. ARNT2 is a highly constrained gene of 717 amino acids with a significant depletion of missense variants in the N-terminus (1-244 aa) and overall fewer loss of function variants in ~282,644 alleles sequenced in gNOMAD. Conclusions: We propose that hypomorphic mutations in ARNT2 could be a potential novel cause of monogenic obesity in humans. Future studies will investigate the molecular mechanisms causing weight dysregulation in patient specific disease relevant hypothalamic neurons. Reference: (1) Turer et al., Dis Model Mech. 2018; 11(12)

Within medical education a reduction in curriculum time for subjects, such as anatomy puts pressure on educators to ensure the same learning outcomes are conveyed in less time. This has the potential to impact negatively on student experience. Near-peer teaching (NPT) is often praised as an effective revision tool, but its use as a frontline teaching resource remains unreported. The study explores the potential for NPT to promote delivery of learning outcomes and maximize student experience within a neuroanatomy module for second year medical students. The study occurred in three educational settings, (1) frontline NPT of cranial nerves, (2) revision session NPT of cranial nerves, and (3) NPT alongside faculty staff in laboratory-based neuroanatomy practical exercises. For the first and second components, knowledge was measured using a pre- and post-session test and student perception was ascertained with a questionnaire. For the third component, student perception was assessed with an end-of-module survey. The results show that overall, NPT was well received by learners. A significant knowledge gain was seen between the pre- and post-session test of the frontline NPT session. The study presents evidence in favor of using NPTs to supplement the delivery of learning outcomes in a time and resource constrained curriculum. In particular, for the effective delivery of frontline material. Anat Sci Educ 0: 1-9. © 2018 American Association of Anatomists.

We report five individuals with loss-of-function of the X-linked AMMECR1: a girl with a balanced X-autosome translocation and inactivation of the normal X-chromosome; two boys with maternally inherited and de novo nonsense variants; and two half-brothers with maternally inherited microdeletion variants. They present with short stature, cardiac and skeletal abnormalities, and hearing loss. Variants of unknown significance in AMMECR1 in four male patients from two families with partially overlapping phenotypes were previously reported. AMMECR1 is coexpressed with genes implicated in cell cycle regulation, five of which were previously associated with growth and bone alterations. Our knockdown of the zebrafish orthologous gene resulted in phenotypes reminiscent of patients' features. The increased transcript and encoded protein levels of AMMECR1L, an AMMECR1 paralog, in the t(X;9) patient's cells indicate a possible partial compensatory mechanism. AMMECR1 and AMMECR1L proteins dimerize and localize to the nucleus as suggested by their nucleic acid-binding RAGNYA folds. Our results suggest that AMMECR1 is potentially involved in cell cycle control and linked to a new syndrome with growth, bone, heart, and kidney alterations with or without elliptocytosis.

Thrombotic microangiopathy is a potentially lethal complication of haematopoietic stem cell (bone marrow) transplantation. The pathophysiology is incompletely understood, although endothelial damage appears to be central. Platelet activation, neutrophil extracellular traps and complement activation appear to play key roles. Diagnosis may be difficult and universally accepted diagnostic criteria are not available. Treatment remains controversial. In some cases, withdrawal of calcineurin inhibitors is adequate. Rituximab and defibrotide also appear to have been used successfully. In severe cases, complement inhibitors such as eculizumab may play a valuable role. Further research is required to define the pathophysiology and determine both robust diagnostic criteria and the optimal treatment.

Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation of DNA (5mC) is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has been delineated. Here, we describe in detail the first Mendelian disorder caused by disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. Here we identify and characterize 11 cases of human TET3 deficiency in 8 families with the common phenotypic features of intellectual disability/global developmental delay, hypotonia, autistic traits, movement disorders, growth abnormalities, and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues with all but one occurring within the catalytic domain and most displaying hypomorphic function in a catalytic activity assay. TET3 deficiency shows substantial phenotypic overlap with other Mendelian disorders of the epigenetic machinery, including intellectual disability and growth abnormalities, underscoring shared disease mechanisms.

Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation (5-methylcytosine [5mC]) of DNA is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has yet been delineated. Here, we describe in detail a Mendelian disorder caused by the disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. We identify and characterize 11 cases of human TET3 deficiency in eight families with the common phenotypic features of intellectual disability and/or global developmental delay; hypotonia; autistic traits; movement disorders; growth abnormalities; and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues; all but one such variant occur within the catalytic domain, and most display hypomorphic function in an assay of catalytic activity. TET3 deficiency and other Mendelian disorders of the epigenetic machinery show substantial phenotypic overlap, including features of intellectual disability and abnormal growth, underscoring shared disease mechanisms.

Motivation Integration of different omics data could markedly help to identify biological signatures, understand the missing heritability of complex diseases and ultimately achieve personalized medicine. Standard regression models used in Genome-Wide Association Studies (GWAS) identify loci with a strong effect size, whereas GWAS meta-analyses are often needed to capture weak loci contributing to the missing heritability. Development of novel machine learning algorithms for merging genotype data with other omics data is highly needed as it could enhance the prioritization of weak loci. Results We developed cNMTF (corrected non-negative matrix tri-factorization), an integrative algorithm based on clustering techniques of biological data. This method assesses the inter-relatedness between genotypes, phenotypes, the damaging effect of the variants and gene networks in order to identify loci-trait associations. cNMTF was used to prioritize genes associated with lipid traits in two population cohorts. We replicated 129 genes reported in GWAS world-wide and provided evidence that supports 85% of our findings (226 out of 265 genes), including recent associations in literature (NLGN1), regulators of lipid metabolism (DAB1) and pleiotropic genes for lipid traits (CARM1). Moreover, cNMTF performed efficiently against strong population structures by accounting for the individuals’ ancestry. As the method is flexible in the incorporation of diverse omics data sources, it can be easily adapted to the user’s research needs. Availability and implementation An R package (cnmtf) is available at https://lgl15.github.io/cnmtf_web/index.html. Supplementary information Supplementary data are available at Bioinformatics online.

SummaryGenetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes critical for an organism’s function will be depleted for such variants in natural populations, while non-essential genes will tolerate their accumulation. However, predicted loss-of-function (pLoF) variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes1. Here, we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence pLoF variants in this cohort after filtering for sequencing and annotation artifacts. Using an improved human mutation rate model, we classify human protein-coding genes along a spectrum representing tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve gene discovery power for both common and rare diseases.

It is important that clinicians are able to adequately assess their level of knowledge and competence in order to be safe practitioners of medicine. The medical literature contains numerous examples of poor self-assessment accuracy amongst medical students over a range of subjects however this ability in neuroanatomy has yet to be observed. Second year medical students attending neuroanatomy revision sessions at the University of Southampton and the competitors of the National Undergraduate Neuroanatomy Competition were asked to rate their level of knowledge in neuroanatomy. The responses from the former group were compared to performance on a ten item multiple choice question examination and the latter group were compared to their performance within the competition. In both cohorts, self-assessments of perceived level of knowledge correlated weakly to their performance in their respective objective knowledge assessments (r = 0.30 and r = 0.44). Within the NUNC, this correlation improved when students were instead asked to rate their performance on a specific examination within the competition (spotter, rS = 0.68; MCQ, rS = 0.58). Despite its inherent difficulty, medical student self-assessment accuracy in neuroanatomy is comparable to other subjects within the medical curriculum. Anat Sci Educ 9: 488-495. © 2016 American Association of Anatomists.

Next-generation sequencing has catapulted healthcare into a revolutionary genomics era. One such technology, whole-exome sequencing, which targets the protein-coding regions of the genome, has proven success in identifying new causal mutations for diseases of previously unknown etiology. With a successful diagnostic rate approaching 25% for rare disease in recent studies, its clinical utility is becoming increasingly popular. However, the interpretation of whole-exome sequencing data requires expertise in genomic informatics and clinical medicine to ensure the accurate and safe reporting of findings back to the bedside. This is challenged by vast amounts of sequencing data harbouring approximately 25 000 variants per sequenced individual. Computational strategies and fastidious filtering frameworks are thus required to extricate candidate variants in a sea of common polymorphisms. Once prioritized, identified variants require intensive scrutiny at a biological level, and require judicious assessment alongside the clinical phenotype. In the final step, all evidence is collated and documented alongside pathogenicity guidelines to produce an exome report that returns to the clinic. This review provides a practical guide for clinicians and genomic informaticians on the clinical application of whole-exome sequencing. We address sequencing capture and methodology, quality control parameters at different stages of sequencing analysis and propose an exome data filtering strategy that includes primary filtering (for the removal of probable benign variants) and secondary filtering for the prioritization of remaining candidates.

Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories.Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software.Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity.Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.

Background Autosomal dominant tubulointerstitial kidney disease (ADTKD) caused by mutations in the UMOD gene (ADTKD-UMOD) is considered rare and often remains unrecognised. We aimed to establish the prevalence of genetic kidney diseases, ADTKD and ADTKD-UMOD in adult chronic kidney disease (CKD) patients, and to investigate characteristic features. Methods We sent questionnaires on family history to all patients with CKD stages 3–5 in our tertiary renal centre to identify patients with inherited renal disease. Details on clinical and family history were obtained from patient interviews and clinical records. Sanger sequencing of the UMOD gene was performed from blood or saliva samples. Results 2027 of 3770 sent questionnaires were returned. 459 patients reported a family history, which was consistent with inherited kidney disease in 217 patients. 182 non-responders with inherited kidney diseases were identified through a database search. Of these 399 individuals, 252 had autosomal dominant polycystic kidney disease (ADPKD), 28 had ADTKD, 25 had Alports, and 44 were unknown, resulting in 11% of CKD 3–5 patients and 19% of end-stage renal disease patients with genetic kidney diseases. Of the unknown, 40 were genotyped, of whom 31 had findings consistent with ADTKD. 30% of unknowns and 39% of unknowns with ADTKD had UMOD mutations. Altogether, 35 individuals from 18 families were found to have ten distinct UMOD mutations (three novel), making up 1% of patients with CKD 3–5, 2% of patients with end-stage renal disease, 9% of inherited kidney diseases and 56% with ADTKD. ADTKD-UMOD was the most common genetic kidney disease after ADPKD with a population prevalence of 9 per million. Less proteinuria and haematuria, but not hyperuricaemia or gout were predictive of ADTKD-UMOD. The main limitations of the study are the single-centre design and a predominantly Caucasian population. Conclusions The prevalence of genetic kidney diseases and ADTKD-UMOD is significantly higher than previously described. Clinical features poorly predicted ADTKD-UMOD, highlighting the need for genetic testing guided by family history alone. Electronic supplementary material The online version of this article (10.1186/s12882-018-1107-y) contains supplementary material, which is available to authorized users.

Near-peer teaching is used in anatomy education because of its benefits to the learner, teacher and faculty members. Despite the range of reports focusing on the learner, the advantages for the teacher, which are thought to include communication skills, subject knowledge and employability, are only beginning to be explored.A questionnaire was distributed to the teachers involved in anatomy near-peer teaching at the University of Southampton and Brighton and Sussex Medical School (BSMS). This questionnaire was designed using a rating scale of 0-10 to assess teacher perspectives on their level of knowledge, teaching skills and enjoyment of teaching. Free-text responses determined the teachers' motivation and perceived benefits from the teaching.Twenty-eight questionnaires were gathered (54.9% response rate), including 20 from Southampton and eight from BSMS. Long-term knowledge retention and better understanding of the material were rated 8.1 and 7.9 out of 10, respectively. Eight responses were from currently practising doctors, who rated how much they now use their teaching skills as doctors as 8.9 out of 10. Of the eight doctors, seven gained points for their foundation programme applications as a direct result of near-peer teaching. The most common motivator for engaging in teaching was to improve subject matter knowledge and the most common benefit was improved communication skills. There are numerous advantages to being a near-peer teacher in medical school DISCUSSION: There are numerous advantages to being a near-peer teacher in medical school, which include knowledge improvement, transferrable professional skills and employability. These initial results support the hypothesised benefits to the teachers and provide a foundation for further longitudinal studies.

Background Multiple genes underlying focal segmental glomerulosclerosis (FSGS) and/or steroid-resistant nephrotic syndrome (SRNS) have been identified, with the recent inclusion of collagen IV mutations responsible for Alport disease (AD) or thin basement membrane nephropathy (TBMN). We aimed to investigate the distribution of gene mutations in adult patients with primary FSGS/SRNS by targeted next generation sequencing (NGS). Methods Eighty-one adults from 76 families were recruited; 24 families had a history of renal disease. A targeted NGS panel was designed and applied, covering 39 genes implicated in FSGS/SRNS including COL4A3-5. Results Confirmed pathogenic mutations were found in 10 patients (6 with family history) from 9 families (diagnostic rate 12%). Probably pathogenic mutations were identified in an additional six patients (combined diagnostic rate 20%). Definitely pathogenic mutations were identified in 22% of patients with family history and 10% without. Mutations in COL4A3-5 were present in eight patients from six families, representing 56% of definitely pathogenic mutations, and establishing a diagnosis of AD in six patients and TBMN in two patients. Collagen mutations were identified in 38% of families with familial FSGS, and 3% with sporadic FSGS, with over half the mutations occurring in COL4A5. Patients with collagen mutations were younger at presentation and more likely to have family history, haematuria and glomerular basement membrane abnormalities. Conclusions We show that collagen IV mutations, including COL4A5, frequently underlie FSGS and should be considered, particularly with a positive family history. Targeted NGS improves diagnostic efficiency by investigating many candidate genes in parallel.

CBL is a tumour suppressor gene on chromosome 11 encoding a multivalent adaptor protein with E3 ubiquitin ligase activity. Germline CBL mutations are dominant, with pathogenic de novo mutations reported that can phenotypically overlap Noonan syndrome.1 Some patients with CBL mutations go on to develop juvenile myelomonocytic leukaemia (JMML), an aggressive malignancy that usually necessitates bone marrow transplantation. Using whole exome sequencing methods, we identified a known mutation in CBL in a 4-year-old Caucasian boy with atypical haemolytic uraemic syndrome (aHUS), moyamoya phenomenon and dysmorphology consistent with a mild Noonan-like phenotype. Exome data revealed loss of heterozygosity across chromosome 11q consistent with JMML but in the absence of clinical leukaemia. Our finding challenges conventional clinical diagnostics since we have identified a pathogenic variant in the CBL gene previously only ascertained in children presenting with leukaemia. The increasing affordability of expansive sequencing is likely to increase the scope of clinical profiles observed for previously identified pathogenic variants and calls into question the interpretability and indications for clinical management.

Background: Neurodevelopmental disorders have challenged clinical genetics for decades, with over 700 genes implicated and many whose function remains unknown. The application of whole-exome sequencing is proving pivotal in closing the genotype/phenotype gap through the discovery of new genes and variants that help to unravel the pathogenic mechanisms driving neuropathogenesis. One such discovery includes TRIO, a gene recently implicated in neurodevelopmental delay. Trio is a Dbl family guanine nucleotide exchange factor (GEF) and a major regulator of neuronal development, controlling actin cytoskeleton dynamics by activating the GTPase Rac1.Methods: Whole-exome sequencing was undertaken on a family presenting with global developmental delay, microcephaly and mild dysmorphism. Father/daughter exome analysis was performed, followed by confirmatory Sanger sequencing and segregation analysis on four individuals. Three further patients were recruited through the deciphering developmental disorders (DDD) study. Functional studies were undertaken using patient-specific Trio protein mutations.Results: We identified a frameshift deletion in TRIO that segregated autosomal dominantly. By scrutinising data from DDD, we further identified three unrelated children with a similar phenotype who harboured de novo missense mutations in TRIO. Biochemical studies demonstrated that in three out of four families, the Trio mutations led to a markedly reduced Rac1 activation.Conclusions: We describe an inherited global developmental delay phenotype associated with a frameshift deletion in TRIO. Additionally, we identify pathogenic de novo missense mutations in TRIO associated with the same consistent phenotype, intellectual disability, microcephaly and dysmorphism with striking digital features. We further functionally validate the importance of the GEF domain in Trio protein function. Our study demonstrates how genomic technologies are yet again proving prolific in diagnosing and advancing the understanding of neurodevelopmental disorders.

Nonsyndromic Cleft Lip and/or Palate (NSCLP) is regarded as a multifactorial condition in which clefting is an isolated phenotype, distinguished from the largely monogenic, syndromic forms which include clefts among a spectrum of phenotypes. Nonsyndromic clefting has been shown to arise through complex interactions between genetic and environmental factors. However, there is increasing evidence that the broad NSCLP classification may include a proportion of cases showing familial patterns of inheritance and contain highly penetrant deleterious variation in specific genes. Through exome sequencing of multi-case families ascertained in Bogota, Colombia, we identify 28 non-synonymous single nucleotide variants that are considered damaging by at least one predictive score. We discuss the functional impact of candidate variants identified. In one family we find a coding variant in the MSX1 gene which is predicted damaging by multiple scores. This variant is in exon 2, a highly conserved region of the gene. Previous sequencing has suggested that mutations in MSX1 may account for ~2% of NSCLP. Our analysis further supports evidence that a proportion of NSCLP cases arise through monogenic coding mutations, though further work is required to unravel the complex interplay of genetics and environment involved in facial clefting.

This report illustrates the difficulties in diagnosing complex cases and demonstrates how whole exome sequencing can resolve complex phenotypes.

The analysis of linkage disequilibrium (LD) underpins the development of effective genotyping technologies, trait mapping and understanding of biological mechanisms such as those driving recombination and the impact of selection. We apply the Malécot-Morton model of LD to create additive LD maps which describe the high-resolution LD landscape of commercial chickens. We investigated LD in chickens (Gallus gallus) at the highest resolution to date for broiler, white egg and brown egg layer commercial lines. There is minimal concordance between breeds of fine scale LD patterns (correlation coefficient < 0.21), and even between discrete broiler lines. Regions of LD breakdown, which may align with recombination hotspots, are enriched near CpG islands and transcription start sites (p < 2.2x10-16), consistent with recent evidence described in finches, but concordance in hotspot locations between commercial breeds is only marginally greater than random. As in other birds functional elements in the chicken genome are associated with recombination, but, unlike evidence from other bird species, the LD landscape is not stable in the populations studied. The development of optimal genotyping panels for genome-led selection programmes will depend on careful analysis of the LD structure of each line of interest. Further study is required to fully elucidate the mechanisms underlying highly divergent LD patterns found in commercial chickens.

It is important that clinicians are able to adequately assess their level of knowledge and competence in order to be safe practitioners of medicine. The medical literature contains numerous examples of poor self-assessment accuracy amongst medical students over a range of subjects however this ability in neuroanatomy has yet to be observed. Second year medical students attending neuroanatomy revision sessions at the University of Southampton and the competitors of the National Undergraduate Neuroanatomy Competition were asked to rate their level of knowledge in neuroanatomy. The responses from the former group were compared to performance on a ten item multiple choice question examination and the latter group were compared to their performance within the competition. In both cohorts, self-assessments of perceived level of knowledge correlated weakly to their performance in their respective objective knowledge assessments (r = 0.30 and r = 0.44). Within the NUNC, this correlation improved when students were instead asked to rate their performance on a specific examination within the competition (spotter, rS = 0.68; MCQ, rS = 0.58). Despite its inherent difficulty, medical student self-assessment accuracy in neuroanatomy is comparable to other subjects within the medical curriculum. Anat Sci Educ 9: 488-495. © 2016 American Association of Anatomists.

This article was migrated. The article was marked as recommended. Near-peer teaching (NPT) is becoming increasing popular in medical education. The rationale and benefits of introducing such programs have been well documented and are usually described in terms of their advantages to the teacher, students and faculty. As a team that have successfully introduced two NPT anatomy programs in the last six years at the University of Southampton, we have taken a largely evidenced based approach in offering 10 considerations to ensure the implementation of a sustainable and effective NPT program in anatomical sciences. We have highlighted important aspects of NPT that will help maximise the benefit of such programs and emphasised particular areas where careful thought is necessary. We conclude that to safeguard sustainability and consistency of any given NPT program, faculty and student partnership is required, as is the maintenance of quality control and evaluative techniques.