The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

Germline homozygous missense DEPDC5 variants cause severe refractory early-onset epilepsy, macrocephaly and bilateral polymicrogyria.

DEPDC5 (DEP Domain-Containing Protein 5) encodes an inhibitory component of the mammalian target of rapamycin (mTOR) pathway and is commonly implicated in sporadic and familial focal epilepsies, both non-lesional and in association with focal cortical dysplasia. Germline pathogenic variants are typically heterozygous and inactivating. We describe a novel phenotype caused by germline biallelic missense variants in DEPDC5. Cases were identified clinically. Available records, including magnetic resonance imaging and electroencephalography, were reviewed. Genetic testing was performed by whole exome and whole-genome sequencing and cascade screening. In addition, immunohistochemistry was performed on skin biopsy. The phenotype was identified in nine children, eight of which are described in detail herein. Six of the children were of Irish Traveller, two of Tunisian and one of Lebanese origin. The Irish Traveller children shared the same DEPDC5 germline homozygous missense variant (p.Thr337Arg), whereas the Lebanese and Tunisian children shared a different germline homozygous variant (p.Arg806Cys). Consistent phenotypic features included extensive bilateral polymicrogyria, congenital macrocephaly and early-onset refractory epilepsy, in keeping with other mTOR-opathies. Eye and cardiac involvement and severe neutropenia were also observed in one or more patients. Five of the children died in infancy or childhood; the other four are currently aged between 5 months and 6 years. Skin biopsy immunohistochemistry was supportive of hyperactivation of the mTOR pathway. The clinical, histopathological and genetic evidence supports a causal role for the homozygous DEPDC5 variants, expanding our understanding of the biology of this gene.

Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Incomplete partition type II in its various manifestations: isolated, in association with EVA, syndromic, and beyond; a multicentre international study.

PURPOSE: Incomplete partition type II (IP-II) is characterized by specific histological features and radiological appearance. It may occur in isolation or in association with an enlarged vestibular aqueduct (EVA). Among those with IP-II and EVA, a subset has a diagnosis of Pendred syndrome. This study aimed to explore the prevalence of isolated IP-II, IP-II with EVA, and cases with a genetic or syndromic basis in our cohort. METHODS: From a large, multicentre database of dysplastic cochleae (446 patients, 892 temporal bones), those with imaging features of IP-II were examined in detail, including whether there was a genetic or syndromic association. RESULTS: A total of 78 patients with IP-II were identified. Among these, 55 patients had bilateral IP-II and EVA (only 12 with typical Mondini triad), 8 with bilateral IP-II and normal VA, 2 with bilateral IP-II and unilateral EVA, and 13 with unilateral IP-II (9 with unilateral EVA). Among the group with bilateral IP-II and bilateral EVA in whom genetic analysis was available, 14 out of 29 (48%) had SLC26A4 mutations and a diagnosis of Pendred syndrome, 1 had a FOXI1 mutation, and a few other genetic abnormalities; none had KCNJ10 pathogenic variants. CONCLUSION: Bilateral IP-II-bilateral EVA may be seen in the context of Pendred syndrome (SLC26A4 or FOXI1 mutations) but, in the majority of our cohort, no genetic abnormalities were found, suggesting the possibility of unknown genetic associations. IP-II in isolation (without EVA) is favored to be genetic when bilateral, although the cause is often unknown.

Bi-allelic Variants in TKFC Encoding Triokinase/FMN Cyclase Are Associated with Cataracts and Multisystem Disease.

We report an inborn error of metabolism caused by TKFC deficiency in two unrelated families. Rapid trio genome sequencing in family 1 and exome sequencing in family 2 excluded known genetic etiologies, and further variant analysis identified rare homozygous variants in TKFC. TKFC encodes a bifunctional enzyme involved in fructose metabolism through its glyceraldehyde kinase activity and in the generation of riboflavin cyclic 4',5'-phosphate (cyclic FMN) through an FMN lyase domain. The TKFC homozygous variants reported here are located within the FMN lyase domain. Functional assays in yeast support the deleterious effect of these variants on protein function. Shared phenotypes between affected individuals with TKFC deficiency include cataracts and developmental delay, associated with cerebellar hypoplasia in one case. Further complications observed in two affected individuals included liver dysfunction and microcytic anemia, while one had fatal cardiomyopathy with lactic acidosis following a febrile illness. We postulate that deficiency of TKFC causes disruption of endogenous fructose metabolism leading to generation of by-products that can cause cataract. In line with this, an affected individual had mildly elevated urinary galactitol, which has been linked to cataract development in the galactosemias. Further, in light of a previously reported role of TKFC in regulating innate antiviral immunity through suppression of MDA5, we speculate that deficiency of TKFC leads to impaired innate immunity in response to viral illness, which may explain the fatal illness observed in the most severely affected individual.

Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders.

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.

A novel ELP1 mutation impairs the function of the Elongator complex and causes a severe neurodevelopmental phenotype.

BACKGROUND: Neurodevelopmental disorders (NDDs) are heterogeneous, debilitating conditions that include motor and cognitive disability and social deficits. The genetic factors underlying the complex phenotype of NDDs remain to be elucidated. Accumulating evidence suggest that the Elongator complex plays a role in NDDs, given that patient-derived mutations in its ELP2, ELP3, ELP4 and ELP6 subunits have been associated with these disorders. Pathogenic variants in its largest subunit ELP1 have been previously found in familial dysautonomia and medulloblastoma, with no link to NDDs affecting primarily the central nervous system. METHODS: Clinical investigation included patient history and physical, neurological and magnetic resonance imaging (MRI) examination. A novel homozygous likely pathogenic ELP1 variant was identified by whole-genome sequencing. Functional studies included in silico analysis of the mutated ELP1 in the context of the holo-complex, production and purification of the ELP1 harbouring the identified mutation and in vitro analyses using microscale thermophoresis for tRNA binding assay and acetyl-CoA hydrolysis assay. Patient fibroblasts were harvested for tRNA modification analysis using HPLC coupled to mass spectrometry. RESULTS: We report a novel missense mutation in the ELP1 identified in two siblings with intellectual disability and global developmental delay. We show that the mutation perturbs the ability of ELP123 to bind tRNAs and compromises the function of the Elongator in vitro and in human cells. CONCLUSION: Our study expands the mutational spectrum of ELP1 and its association with different neurodevelopmental conditions and provides a specific target for genetic counselling.

The spectrum of cochlear malformations in CHARGE syndrome and insights into the role of the CHD7 gene during embryogenesis of the inner ear.

PURPOSE: We reviewed the genotypes and the imaging appearances of cochleae in CHARGE patients from two large tertiary centres and analysed the observed cochlear anomalies, providing detailed anatomical description and a grading system. The goal was to gain insight into the spectrum of cochlear anomalies in CHARGE syndrome, and thus, in the role of the CHD7 gene in otic vesicle development. METHODS: We retrospectively reviewed CT and/or MR imaging of CHARGE patients referred to our institutions between 2005 and 2022. Cochlear morphology was analysed and, when abnormal, divided into 3 groups in order of progressive severity. Other radiological findings in the temporal bone were also recorded. Comparison with the existing classification system of cochlear malformation was also attempted. RESULTS: Cochlear morphology in our CHARGE cohort ranged from normal to extreme hypoplasia. The most common phenotype was cochlear hypoplasia in which the basal turn was relatively preserved, and the upper turns were underdeveloped. All patients in the cohort had absent or markedly hypoplastic semicircular canals and small, misshapen vestibules. Aside from a stenotic cochlear aperture (fossette) being associated with a hypoplastic or absent cochlear nerve, there was no consistent relationship between cochlear nerve status (normal, hypoplasia, or aplasia) and cochlear morphology. CONCLUSION: Cochlear morphology in CHARGE syndrome is variable. Whenever the cochlea was abnormal, it was almost invariably hypoplastic. This may shed light on the role of CHD7 in cochlear development. Accurate morphological description of the cochlea contributes to proper clinical diagnosis and is important for planning surgical treatment options.

Syndromic Forms of Hyperinsulinaemic Hypoglycaemia-A 15-year follow-up Study.

OBJECTIVE: Hyperinsulinaemic hypoglycaemia (HH) is one of the commonest causes of hypoglycaemia in children. The molecular basis includes defects in pathways that regulate insulin release. Syndromic conditions like Beckwith-Wiedemann (BWS), Kabuki (KS) and Turner (TS) are known to be associated with a higher risk for HH. This systematic review of children with HH referred to a tertiary centre aims at estimating the frequency of a syndromic/multisystem condition to help address stratification of genetic analysis in infants with HH. METHODS: We performed a retrospective study of 69 patients with syndromic features and hypoglycaemia in a specialist centre from 2004 to 2018. RESULTS: Biochemical investigations confirmed HH in all the cases and several genetic diagnoses were established. Responsiveness to medications and the final outcome following medical treatment or surgery were studied. CONCLUSIONS: This study highlights the association of HH with a wide spectrum of syndromic diagnoses and that children with features suggestive of HH-associated syndromes should be monitored for hypoglycaemia. If hypoglycaemia is documented, they should also be screened for possible HH. Our data indicate that most syndromic forms of HH are diazoxide-responsive and that HH resolves over time; however, a significant percentage continues to require medications years after the onset of the disease. Early diagnosis of hyperinsulinism and initiation of treatment is important for preventing hypoglycaemic brain injury and intellectual disability.

Critical Review: Role of Inorganic Nanoparticle Properties on Their Foliar Uptake and in Planta Translocation.

There is increasing pressure on global agricultural systems due to higher food demand, climate change, and environmental concerns. The design of nanostructures is proposed as one of the economically viable technological solutions that can make agrochemical use (fertilizers and pesticides) more efficient through reduced runoff, increased foliar uptake and bioavailability, and decreased environmental impacts. However, gaps in knowledge about the transport of nanoparticles across the leaf surface and their behavior in planta limit the rational design of nanoparticles for foliar delivery with controlled fate and limited risk. Here, the current literature on nano-objects deposited on leaves is reviewed. The different possible foliar routes of uptake (stomata, cuticle, trichomes, hydathodes, necrotic spots) are discussed, along with the paths of translocation, via the phloem, from the leaf to the end sinks (mature and developing tissues, roots, rhizosphere). This review details the interplays between morphological constraints, environmental stimuli, and physical-chemical properties of nanoparticles influencing their fate, transformation, and transport after foliar deposition. A metadata analysis from the existing literature highlighted that plant used for testing nanoparticle fate are most often dicotyledon plants (75%), while monocotyledons (as cereals) are less considered. Correlations on parameters calculated from the literature indicated that nanoparticle dose, size, zeta potential, and affinity to organic phases correlated with leaf-to-sink translocation, demonstrating that targeting nanoparticles to specific plant compartments by design should be achievable. Correlations also showed that time and plant growth seemed to be drivers for in planta mobility, parameters that are largely overlooked in the literature. This review thus highlights the material design opportunities and the knowledge gaps for targeted, stimuli driven deliveries of safe nanomaterials for agriculture.

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease.

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.

PIGT-CDG, a disorder of the glycosylphosphatidylinositol anchor: description of 13 novel patients and expansion of the clinical characteristics.

PURPOSE: To provide a detailed electroclinical description and expand the phenotype of PIGT-CDG, to perform genotype-phenotype correlation, and to investigate the onset and severity of the epilepsy associated with the different genetic subtypes of this rare disorder. Furthermore, to use computer-assisted facial gestalt analysis in PIGT-CDG and to the compare findings with other glycosylphosphatidylinositol (GPI) anchor deficiencies. METHODS: We evaluated 13 children from eight unrelated families with homozygous or compound heterozygous pathogenic variants in PIGT. RESULTS: All patients had hypotonia, severe developmental delay, and epilepsy. Epilepsy onset ranged from first day of life to two years of age. Severity of the seizure disorder varied from treatable seizures to severe neonatal onset epileptic encephalopathies. The facial gestalt of patients resembled that of previously published PIGT patients as they were closest to the center of the PIGT cluster in the clinical face phenotype space and were distinguishable from other gene-specific phenotypes. CONCLUSION: We expand our knowledge of PIGT. Our cases reaffirm that the use of genetic testing is essential for diagnosis in this group of disorders. Finally, we show that computer-assisted facial gestalt analysis accurately assigned PIGT cases to the multiple congenital anomalies-hypotonia-seizures syndrome phenotypic series advocating the additional use of next-generation phenotyping technology.

Rapid Paediatric Sequencing (RaPS): comprehensive real-life workflow for rapid diagnosis of critically ill children.

BACKGROUND: Rare genetic conditions are frequent risk factors for, or direct causes of, paediatric intensive care unit (PICU) admission. Such conditions are frequently suspected but unidentified at PICU admission. Compassionate and effective care is greatly assisted by definitive diagnostic information. There is therefore a need to provide a rapid genetic diagnosis to inform clinical management.To date, whole genome sequencing (WGS) approaches have proved successful in diagnosing a proportion of children with rare diseases, but results may take months to report. Our aim was to develop an end-to-end workflow for the use of rapid WGS for diagnosis in critically ill children in a UK National Health Service (NHS) diagnostic setting. METHODS: We sought to establish a multidisciplinary Rapid Paediatric Sequencing team for case selection, trio WGS, rapid bioinformatics sequence analysis and a phased analysis and reporting system to prioritise genes with a high likelihood of being causal. RESULTS: Trio WGS in 24 critically ill children led to a molecular diagnosis in 10 (42%) through the identification of causative genetic variants. In 3 of these 10 individuals (30%), the diagnostic result had an immediate impact on the individual's clinical management. For the last 14 trios, the shortest time taken to reach a provisional diagnosis was 4 days (median 8.5 days). CONCLUSION: Rapid WGS can be used to diagnose and inform management of critically ill children within the constraints of an NHS clinical diagnostic setting. We provide a robust workflow that will inform and facilitate the rollout of rapid genome sequencing in the NHS and other healthcare systems globally.

Preparing for Otoferlin gene therapy trials: A survey of NHS Paediatric Audiology and Cochlear Implant services on diagnosis and management of Auditory Neuropathy Spectrum Disorder.

OBJECTIVES: Gene therapy for monogenic hearing loss is on the horizon. The first trials in patients with Auditory Neuropathy Spectrum Disorder (ANSD) due to pathogenic variants in the Otoferlin (OTOF) gene will open this year. In the UK, the new NHS Genomic Medicine Service (GMS) offers genetic testing in each child diagnosed with congenital or early onset sensorineural hearing loss. This survey study aims to map preexisting clinical pathways for the diagnosis and management of children with ANSD and identify opportunities for improvement in early identification of OTOF- related ANSD. METHODS: A Google form with 24 questions in English covering the ANSD clinical pathway was developed with clinicians involved in the diagnosis and management ANSD. The survey was disseminated via email to all Lead clinicians of NHS Tertiary Paediatric Audiology and Cochlear Implant Services within the UK. RESULTS: Data was received from 27 (34 %) NHS Tertiary Paediatric Audiology Services and 8 (n = 57 %) Paediatric Cochlear Implant Services. Services follow existing national guidance and provide multidisciplinary care with structured patient pathways for referral, diagnosis, and management of children with ANSD and multidisciplinary input throughout. Clinicians are aware of the genetic causes of ANSD and new processes for genetic testing, but do not uniformly refer children with ANSD for testing for OTOF pathogenic variants. As such, they had difficulty estimating numbers of children with OTOF pathogenic variants under their care. CONCLUSION: Those results highlight the urgency of implementing hearing gene panel sequencing for all children with ANSD to provide opportunities for early diagnosis and candidacy for OTOF gene therapy trials.

Exclusion of WWP1 mutations in a cohort of dystroglycanopathy patients.

INTRODUCTION: Aberrant glycosylation of α-dystroglycan is associated with a subset of clinically heterogeneous muscular dystrophies collectively referred to as the dystroglycanopathies. These autosomal-recessive disorders span a wide spectrum of clinical severity ranging from Walker-Warburg syndrome, with severe brain and eye abnormalities, to mild adult-onset limb-girdle muscular dystrophy. To date, seven causative genes have been identified in the dystroglycanopathies, yet studies have suggested that a significant proportion of patients harbor mutations in novel genes. METHODS: A homozygous missense alteration in the gene encoding ubiquitin ligase WW domain-containing protein 1 (WWP1), has recently been identified in the dystroglycanopathy chicken. We therefore investigated whether mutations in the human ortholog were present in a cohort of 33 dystroglycanopathy patients. RESULTS: No clear pathogenic mutations were identified. CONCLUSION: The present findings indicate that WWP1 is not a common cause of human dystroglycanopathy.

Persistently elevated CK and lysosomal storage myopathy associated with mucolipin 1 defects.

Mucolipidosis type IV is a rare autosomal recessive lysosomal storage disorder caused by bi-allelic pathogenic variants in the gene MCOLN1. This encodes for mucolipin-1 (ML1), an endo-lysosomal transmembrane Ca++ channel involved in vesicular trafficking. Although experimental models suggest that defects in mucolipin-1 can cause muscular dystrophy, putatively due to defective lysosomal-mediated sarcolemma repair, the role of mucolipin-1 in human muscle is still poorly deciphered. Elevation of creatine kinase (CK) had been reported in a few cases in the past but comprehensive descriptions of muscle pathology are lacking. Here we report a 7-year-old boy who underwent muscle biopsy due to persistently elevated CK levels (780-15,000 UI/L). Muscle pathology revealed features of a lysosomal storage myopathy with mild regenerative changes. Next generation sequencing confirmed homozygous nonsense variants in MCOLN1. This is a comprehensive pathological description of ML1-related myopathy, supporting the role of mucolipin-1 in muscle homoeostasis.

Mixed-methods evaluation of the NHS Genomic Medicine Service for paediatric rare diseases: study protocol.

Background: A new nationally commissioned NHS England Genomic Medicine Service (GMS) was recently established to deliver genomic testing with equity of access for patients affected by rare diseases and cancer. The overarching aim of this research is to evaluate the implementation of the GMS during its early years, identify barriers and enablers to successful implementation, and provide recommendations for practice. The focus will be on the use of genomic testing for paediatric rare diseases. Methods: This will be a four-year mixed-methods research programme using clinic observations, interviews and surveys. Study 1 consists of qualitative interviews with designers/implementers of the GMS in Year 1 of the research programme, along with documentary analysis to understand the intended outcomes for the Service. These will be revisited in Year 4 to compare intended outcomes with what happened in practice, and to identify barriers and facilitators that were encountered along the way. Study 2 consists of clinic observations (pre-test counselling and results disclosure) to examine the interaction between health professionals and parents, along with follow-up interviews with both after each observation. Study 3 consists of a longitudinal survey with parents at two timepoints (time of testing and 12 months post-results) along with follow-up interviews, to examine parent-reported experiences and outcomes. Study 4 consists of qualitative interviews and a cross-sectional survey with medical specialists to identify preparedness, facilitators and challenges to mainstreaming genomic testing. The use of theory-based and pre-specified constructs will help generalise the findings and enable integration across the various sub-studies. Dissemination: We will disseminate our results to policymakers as findings emerge, so any suggested changes to service provision can be considered in a timely manner. A workshop with key stakeholders will be held in Year 4 to develop and agree a set of recommendations for practice.

BACKGROUND AND AIMS: Genome sequencing (where a person's entire genetic code is mapped) is set to dramatically transform patient care and medical outcomes. Recently, genome sequencing was introduced as part of routine clinical care in the NHS, through the Genomic Medicine Service (GMS). The aim of this research is to understand how genome sequencing is being delivered in the first few years of the Service, in particular what the barriers and enablers are to successful delivery. The focus of the study will be the use of genome sequencing for children with undiagnosed conditions. STUDY DESIGN: This is a four-year study in which we will conduct: observations of clinic appointments; interviews with policy makers and health professionals designing and implementing the new service; and surveys/interviews with parents of patients undergoing genomic testing. By the end of this study we will have: - a better understanding of the intended vs actual outcomes of the GMS,- insights into what happens during clinical encounters,- understand what the entire testing process is like for parents from being offered genomic testing to receiving their results and beyond, including the clinical as well as emotional and practical outcomes, and- understand how healthcare professionals feel about delivering the GMS, particularly those that are non-genetic specialists, including how prepared they feel to deliver genomic testing. Patient and public involvement: Parents of children who have been through the testing process have helped us design this study. They have inputted into surveys and topic guides, and will be involved throughout the study as members of the advisory team so that we can ensure the findings are used to improve the quality of care patients and families receive. DISSEMINATION: The findings from this research will be shared with organisations such as NHS England and NHS Improvement so that recommendations can be implemented swiftly.

Use of whole genome sequencing to determine genetic basis of suspected mitochondrial disorders: cohort study.

OBJECTIVE: To determine whether whole genome sequencing can be used to define the molecular basis of suspected mitochondrial disease. DESIGN: Cohort study. SETTING: National Health Service, England, including secondary and tertiary care. PARTICIPANTS: 345 patients with suspected mitochondrial disorders recruited to the 100 000 Genomes Project in England between 2015 and 2018. INTERVENTION: Short read whole genome sequencing was performed. Nuclear variants were prioritised on the basis of gene panels chosen according to phenotypes, ClinVar pathogenic/likely pathogenic variants, and the top 10 prioritised variants from Exomiser. Mitochondrial DNA variants were called using an in-house pipeline and compared with a list of pathogenic variants. Copy number variants and short tandem repeats for 13 neurological disorders were also analysed. American College of Medical Genetics guidelines were followed for classification of variants. MAIN OUTCOME MEASURE: Definite or probable genetic diagnosis. RESULTS: A definite or probable genetic diagnosis was identified in 98/319 (31%) families, with an additional 6 (2%) possible diagnoses. Fourteen of the diagnoses (4% of the 319 families) explained only part of the clinical features. A total of 95 different genes were implicated. Of 104 families given a diagnosis, 39 (38%) had a mitochondrial diagnosis and 65 (63%) had a non-mitochondrial diagnosis. CONCLUSION: Whole genome sequencing is a useful diagnostic test in patients with suspected mitochondrial disorders, yielding a diagnosis in a further 31% after exclusion of common causes. Most diagnoses were non-mitochondrial disorders and included developmental disorders with intellectual disability, epileptic encephalopathies, other metabolic disorders, cardiomyopathies, and leukodystrophies. These would have been missed if a targeted approach was taken, and some have specific treatments.

Delivering genome sequencing for rapid genetic diagnosis in critically ill children: parent and professional views, experiences and challenges.

Rapid genomic sequencing (RGS) is increasingly being used in the care of critically ill children. Here we describe a qualitative study exploring parent and professional perspectives around the usefulness of this test, the potential for unintended harms and the challenges for delivering a wider clinical service. The Rapid Paediatric Sequencing (RaPS) study offered trio RGS for diagnosis of critically ill children with a likely monogenic disorder. Main and actionable secondary findings were reported. Semi-structured interviews were conducted with parents of children offered RGS (n = 11) and professionals (genetic clinicians, non-genetic clinicians, scientists and consenters) (n = 19) by telephone (parents n = 10/professionals n = 1) or face-to-face (parents n = 1/professionals n = 18). We found that participants held largely positive views about RGS, describing clinical and emotional benefits from the opportunity to obtain a rapid diagnosis. Parental stress surrounding their child's illness complicates decision making. Parental concerns are heightened when offered RGS and while waiting for results. The importance of multidisciplinary team working to enable efficient delivery of a rapid service was emphasised. Our findings give insight into the perceived value of RGS for critically ill children. Careful pre-test counselling is needed to support informed parental decision making. Many parents would benefit from additional support while waiting for results. Education of mainstream clinicians is required to facilitate clinical implementation.

Protein coating composition targets nanoparticles to leaf stomata and trichomes.

Plant nanobiotechnology has the potential to revolutionize agriculture. However, the lack of effective methods to deliver nanoparticles (NPs) to the precise locations in plants where they are needed impedes these technological innovations. Here, model gold nanoparticles (AuNP) were coated with citrate, bovine serum albumin (BSA) as a protein control, or LM6-M, an antibody with an affinity for functional groups unique to stomata on leaf surfaces to deliver the AuNPs to stomata. One-month-old Vicia faba leaves were exposed via drop deposition to aqueous suspensions of LM6-M-coated AuNPs and allowed to air dry. After rinsing, Au distribution on the leaf surface was investigated by enhanced dark-field microscopy and X-ray fluorescence mapping. While citrate-coated AuNPs randomly covered the plant leaves, LM6M-AuNPs strongly adhered to the stomata and remained on the leaf surface after rinsing, and BSA-AuNPs specifically targeted trichome hairs. To the authors' knowledge, this is the first report of active targeting of live leaf structures using NPs coated with molecular recognition molecules. This proof-of-concept study provides a strategy for future targeted nanopesticide delivery research.