Sheffield Children's NHS Foundation Trust

The Ehlers-Danlos syndromes (EDS) are a clinically and genetically heterogeneous group of heritable connective tissue disorders (HCTDs) characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. Over the past two decades, the Villefranche Nosology, which delineated six subtypes, has been widely used as the standard for clinical diagnosis of EDS. For most of these subtypes, mutations had been identified in collagen-encoding genes, or in genes encoding collagen-modifying enzymes. Since its publication in 1998, a whole spectrum of novel EDS subtypes has been described, and mutations have been identified in an array of novel genes. The International EDS Consortium proposes a revised EDS classification, which recognizes 13 subtypes. For each of the subtypes, we propose a set of clinical criteria that are suggestive for the diagnosis. However, in view of the vast genetic heterogeneity and phenotypic variability of the EDS subtypes, and the clinical overlap between EDS subtypes, but also with other HCTDs, the definite diagnosis of all EDS subtypes, except for the hypermobile type, relies on molecular confirmation with identification of (a) causative genetic variant(s). We also revised the clinical criteria for hypermobile EDS in order to allow for a better distinction from other joint hypermobility disorders. To satisfy research needs, we also propose a pathogenetic scheme, that regroups EDS subtypes for which the causative proteins function within the same pathway. We hope that the revised International EDS Classification will serve as a new standard for the diagnosis of EDS and will provide a framework for future research purposes. © 2017 Wiley Periodicals, Inc.

The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results. Low read depth sequencing of whole genomes and high read depth exomes of nearly 10,000 extensively phenotyped individuals are combined to help characterize novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with lipid-related traits; in addition to describing population structure and providing functional annotation of rare and low-frequency variants the authors use the data to estimate the benefits of sequencing for association studies. This paper, combining data and initial findings from the different arms of the UK10K project, describes insights from low-read-depth sequencing of whole genomes or high-read-depth exome sequencing of nearly 10,000 individuals sampled from a range of disease collections, as well as participants from healthy population based cohorts. The authors characterize novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with lipid-related traits. In addition to describing population structure and providing functional annotation of rare and low frequency variants, they use the data to estimate the benefits of sequencing for association studies.

Abstract The genetic make-up of an individual contributes to the susceptibility and response to viral infection. Although environmental, clinical and social factors have a role in the chance of exposure to SARS-CoV-2 and the severity of COVID-19 1,2 , host genetics may also be important. Identifying host-specific genetic factors may reveal biological mechanisms of therapeutic relevance and clarify causal relationships of modifiable environmental risk factors for SARS-CoV-2 infection and outcomes. We formed a global network of researchers to investigate the role of human genetics in SARS-CoV-2 infection and COVID-19 severity. Here we describe the results of three genome-wide association meta-analyses that consist of up to 49,562 patients with COVID-19 from 46 studies across 19 countries. We report 13 genome-wide significant loci that are associated with SARS-CoV-2 infection or severe manifestations of COVID-19. Several of these loci correspond to previously documented associations to lung or autoimmune and inflammatory diseases 3–7 . They also represent potentially actionable mechanisms in response to infection. Mendelian randomization analyses support a causal role for smoking and body-mass index for severe COVID-19 although not for type II diabetes. The identification of novel host genetic factors associated with COVID-19 was made possible by the community of human genetics researchers coming together to prioritize the sharing of data, results, resources and analytical frameworks. This working model of international collaboration underscores what is possible for future genetic discoveries in emerging pandemics, or indeed for any complex human disease.

<h3>Introduction</h3> The forearm is the most common fracture location in children, with an increasing incidence. Displaced forearm shaft fractures have traditionally been treated with closed reduction and cast immobilisation. Diaphyseal fractures in children have poor remodelling capacity. Malunion can cause permanent cosmetic and functional disability. Internal fixation with flexible intramedullary nails has gained increasing popularity, without evidence of a better outcome compared with closed reduction and cast immobilisation. <h3>Method and analysis</h3> This is a multicentre, randomised superiority trial comparing closed reduction and cast immobilisation to flexible intramedullary nails in children aged 7–12 years with &gt;10° of angulation and/or &gt;10 mm of shortening in displaced both bone forearm shaft fractures (AO-paediatric classification: 22D/2.1–5.2). A total of 78 patients with minimum 2 years of expected growth left are randomised in 1:1 ratio to either treatment group. The study has a parallel non-randomised patient preference arm. Both treatments are performed under general anaesthesia. In the cast group a long arm cast is applied for 6 weeks. The flexible intramedullary nail group is immobilised in a collar and cuff sling for 4 weeks. Data are collected at baseline and at each follow-up until 1 year. Primary outcome is (1) PROMIS paediatric upper extremity and (2) forearm pronation-supination range of motion at 1-year follow-up. Secondary outcomes are Quick DASH, Paediatric Pain Questionnaire, Cosmetic Visual Analogue Scale, wrist and elbow range of motion as well as any complications and costs of treatment. We hypothesise that flexible intramedullary nailing results in a superior outcome. <h3>Ethics and dissemination</h3> We have received ethical board approval (number: 78/1801/2020) and permissions to conduct the study from all five participating university hospitals. Informed consent is obtained from the parent(s). Results will be disseminated in peer-reviewed publications. <h3>Trial registration number</h3> NCT04664517.

BACKGROUND: Fetal structural anomalies, which are detected by ultrasonography, have a range of genetic causes, including chromosomal aneuploidy, copy number variations (CNVs; which are detectable by chromosomal microarrays), and pathogenic sequence variants in developmental genes. Testing for aneuploidy and CNVs is routine during the investigation of fetal structural anomalies, but there is little information on the clinical usefulness of genome-wide next-generation sequencing in the prenatal setting. We therefore aimed to evaluate the proportion of fetuses with structural abnormalities that had identifiable variants in genes associated with developmental disorders when assessed with whole-exome sequencing (WES). METHODS: In this prospective cohort study, two groups in Birmingham and London recruited patients from 34 fetal medicine units in England and Scotland. We used whole-exome sequencing (WES) to evaluate the presence of genetic variants in developmental disorder genes (diagnostic genetic variants) in a cohort of fetuses with structural anomalies and samples from their parents, after exclusion of aneuploidy and large CNVs. Women were eligible for inclusion if they were undergoing invasive testing for identified nuchal translucency or structural anomalies in their fetus, as detected by ultrasound after 11 weeks of gestation. The partners of these women also had to consent to participate. Sequencing results were interpreted with a targeted virtual gene panel for developmental disorders that comprised 1628 genes. Genetic results related to fetal structural anomaly phenotypes were then validated and reported postnatally. The primary endpoint, which was assessed in all fetuses, was the detection of diagnostic genetic variants considered to have caused the fetal developmental anomaly. FINDINGS: The cohort was recruited between Oct 22, 2014, and June 29, 2017, and clinical data were collected until March 31, 2018. After exclusion of fetuses with aneuploidy and CNVs, 610 fetuses with structural anomalies and 1202 matched parental samples (analysed as 596 fetus-parental trios, including two sets of twins, and 14 fetus-parent dyads) were analysed by WES. After bioinformatic filtering and prioritisation according to allele frequency and effect on protein and inheritance pattern, 321 genetic variants (representing 255 potential diagnoses) were selected as potentially pathogenic genetic variants (diagnostic genetic variants), and these variants were reviewed by a multidisciplinary clinical review panel. A diagnostic genetic variant was identified in 52 (8·5%; 95% CI 6·4-11·0) of 610 fetuses assessed and an additional 24 (3·9%) fetuses had a variant of uncertain significance that had potential clinical usefulness. Detection of diagnostic genetic variants enabled us to distinguish between syndromic and non-syndromic fetal anomalies (eg, congenital heart disease only vs a syndrome with congenital heart disease and learning disability). Diagnostic genetic variants were present in 22 (15·4%) of 143 fetuses with multisystem anomalies (ie, more than one fetal structural anomaly), nine (11·1%) of 81 fetuses with cardiac anomalies, and ten (15·4%) of 65 fetuses with skeletal anomalies; these phenotypes were most commonly associated with diagnostic variants. However, diagnostic genetic variants were least common in fetuses with isolated increased nuchal translucency (≥4·0 mm) in the first trimester (in three [3·2%] of 93 fetuses). INTERPRETATION: WES facilitates genetic diagnosis of fetal structural anomalies, which enables more accurate predictions of fetal prognosis and risk of recurrence in future pregnancies. However, the overall detection of diagnostic genetic variants in a prospectively ascertained cohort with a broad range of fetal structural anomalies is lower than that suggested by previous smaller-scale studies of fewer phenotypes. WES improved the identification of genetic disorders in fetuses with structural abnormalities; however, before clinical implementation, careful consideration should be given to case selection to maximise clinical usefulness. FUNDING: UK Department of Health and Social Care and The Wellcome Trust.

Lung infections with Mycobacterium abscessus, a species of multidrug-resistant nontuberculous mycobacteria, are emerging as an important global threat to individuals with cystic fibrosis (CF), in whom M. abscessus accelerates inflammatory lung damage, leading to increased morbidity and mortality. Previously, M. abscessus was thought to be independently acquired by susceptible individuals from the environment. However, using whole-genome analysis of a global collection of clinical isolates, we show that the majority of M. abscessus infections are acquired through transmission, potentially via fomites and aerosols, of recently emerged dominant circulating clones that have spread globally. We demonstrate that these clones are associated with worse clinical outcomes, show increased virulence in cell-based and mouse infection models, and thus represent an urgent international infection challenge.

Respiration rate is an important indicator of a person's health, and thus it is monitored when performing clinical evaluations. There are different approaches for respiration monitoring, but generally they can be classed as contact or noncontact. For contact methods, the sensing device (or part of the instrument containing it) is attached to the subject's body. For noncontact approaches the monitoring is performed by an instrument that does not make any contact with the subject. In this article a review of respiration monitoring approaches (both contact and noncontact) is provided. Concerns related to the patient's recording comfort, recording hygiene, and the accuracy of respiration rate monitoring have resulted in the development of a number of noncontact respiration monitoring approaches. A description of thermal imaging based and vision based noncontact respiration monitoring approaches we are currently developing is provided.

Imputing genotypes from reference panels created by whole-genome sequencing (WGS) provides a cost-effective strategy for augmenting the single-nucleotide polymorphism (SNP) content of genome-wide arrays. The UK10K Cohorts project has generated a data set of 3,781 whole genomes sequenced at low depth (average 7x), aiming to exhaustively characterize genetic variation down to 0.1% minor allele frequency in the British population. Here we demonstrate the value of this resource for improving imputation accuracy at rare and low-frequency variants in both a UK and an Italian population. We show that large increases in imputation accuracy can be achieved by re-phasing WGS reference panels after initial genotype calling. We also present a method for combining WGS panels to improve variant coverage and downstream imputation accuracy, which we illustrate by integrating 7,562 WGS haplotypes from the UK10K project with 2,184 haplotypes from the 1000 Genomes Project. Finally, we introduce a novel approximation that maintains speed without sacrificing imputation accuracy for rare variants.

BACKGROUND: Measurement of lung volumes across the life course is critical to the diagnosis and management of lung disease. The aim of the study was to use the Global Lung Function Initiative methodology to develop all-age multi-ethnic reference equations for lung volume indices determined using body plethysmography and gas dilution techniques. METHODS: Static lung volume data from body plethysmography and gas dilution techniques from individual, healthy participants were collated. Reference equations were derived using the LMS (lambda-mu-sigma) method and the generalised additive models of location shape and scale programme in R. The impact of measurement technique, equipment type and being overweight or obese on the derived lung volume reference ranges was assessed. RESULTS: Data from 17 centres were submitted and reference equations were derived from 7190 observations from participants of European ancestry between the ages of 5 and 80 years. Data from non-European ancestry populations were insufficient to develop multi-ethnic equations. Measurements of functional residual capacity (FRC) collected using plethysmography and dilution techniques showed physiologically insignificant differences and were combined. Sex-specific reference equations including height and age were developed for total lung capacity (TLC), FRC, residual volume (RV), inspiratory capacity, vital capacity, expiratory reserve volume and RV/TLC. The derived equations were similar to previously published equations for FRC and TLC, with closer agreement during childhood and adolescence than in adulthood. CONCLUSIONS: Global Lung Function Initiative reference equations for lung volumes provide a generalisable standard for reporting and interpretation of lung volumes measurements in individuals of European ancestry.

OBJECTIVE: To compare the long-term self-esteem and social function outcomes of individuals with untreated and treated ADHD across childhood, adolescence, and adulthood. METHOD: A systematic search of 12 databases was performed to identify peer-reviewed, primary research articles, published January 1980 to December 2011, reporting long-term self-esteem and/or social function outcomes (≥2 years; life consequences distinct from symptoms) of individuals with untreated or treated ADHD. RESULTS: Overall, 127 studies reported 150 outcomes. Most outcomes were poorer in individuals with untreated ADHD versus non-ADHD controls (57% [13/23] for self-esteem; 73% [52/71] for social function). A beneficial response to treatment (pharmacological, nonpharmacological, and multimodal treatments) was reported for the majority of self-esteem (89% [8/9]) and social function (77% [17/22]) outcomes. CONCLUSION: Untreated ADHD was associated with poorer long-term self-esteem and social function outcomes compared with non-ADHD controls. Treatment for ADHD was associated with improvement in outcomes; however, further long-term outcome studies are needed.

Previous studies have failed to identify mutations in the Wilson's disease gene ATP7B in a significant number of clinically diagnosed cases. This has led to concerns about genetic heterogeneity for this condition but also suggested the presence of unusual mutational mechanisms. We now present our findings in 181 patients from the United Kingdom with clinically and biochemically confirmed Wilson's disease. A total of 116 different ATP7B mutations were detected, 32 of which are novel. The overall mutation detection frequency was 98%. The likelihood of mutations in genes other than ATP7B causing a Wilson's disease phenotype is therefore very low. We report the first cases with Wilson's disease due to segmental uniparental isodisomy as well as three patients with three ATP7B mutations and three families with Wilson's disease in two consecutive generations. We determined the genetic prevalence of Wilson's disease in the United Kingdom by sequencing the entire coding region and adjacent splice sites of ATP7B in 1000 control subjects. The frequency of all single nucleotide variants with in silico evidence of pathogenicity (Class 1 variant) was 0.056 or 0.040 if only those single nucleotide variants that had previously been reported as mutations in patients with Wilson's disease were included in the analysis (Class 2 variant). The frequency of heterozygote, putative or definite disease-associated ATP7B mutations was therefore considerably higher than the previously reported occurrence of 1:90 (or 0.011) for heterozygote ATP7B mutation carriers in the general population (P < 2.2 × 10(-16) for Class 1 variants or P < 5 × 10(-11) for Class 2 variants only). Subsequent exclusion of four Class 2 variants without additional in silico evidence of pathogenicity led to a further reduction of the mutation frequency to 0.024. Using this most conservative approach, the calculated frequency of individuals predicted to carry two mutant pathogenic ATP7B alleles is 1:7026 and thus still considerably higher than the typically reported prevalence of Wilson's disease of 1:30 000 (P = 0.00093). Our study provides strong evidence for monogenic inheritance of Wilson's disease. It also has major implications for ATP7B analysis in clinical practice, namely the need to consider unusual genetic mechanisms such as uniparental disomy or the possible presence of three ATP7B mutations. The marked discrepancy between the genetic prevalence and the number of clinically diagnosed cases of Wilson's disease may be due to both reduced penetrance of ATP7B mutations and failure to diagnose patients with this eminently treatable disorder.

The guideline group was selected to be representative of UK-based medical experts. MEDLINE and EMBASE were searched systematically for publications in English from 2002 using the key word Willebrand. The writing group produced the draft guideline, which was subsequently reviewed by the A United Kingdom Haemophilia Centre Doctors Organization (UKHCDO) advisory committee, a British Committee for Standards in Haematology (BCSH) sounding board of approximately 50 UK haematologists, and the BCSH executive; comments were incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found in at http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMENDATION/43_GRADE.html. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of patients with von Willebrand disease. This is a single guideline replacing two separate guidelines on diagnosis and management respectively, published in 2004 (Laffan et al, 2004; Pasi et al, 2004). Where there has been no significant change in understanding or practice, the reader is referred to the earlier guidelines. The principal changes have been increased understanding of the genetic basis of von Willebrand disease, a relaxation of definition and a focus on how laboratory tests can guide management. Von Willebrand factor (VWF) is a large and complex plasma glycoprotein that is essential for normal haemostasis. It is well recognized that deficiency of VWF results in a bleeding disorder that varies in severity according to the degree of deficiency and the specific characteristics of the molecule and which may have features of both primary and secondary haemostatic defects. The complex structure of the protein and the wide range of plasma levels encountered in the population make laboratory assessment and diagnosis a challenging proposition. Since the last guidelines by this group (Laffan et al, 2004; Pasi et al, 2004), there have been considerable advances in understanding the genetics, function and clinical correlates of VWF, which have been incorporated into this revised and unified document. Here we define von Willebrand disease (VWD) as a bleeding disorder that is predominantly attributable to reduced levels of VWF activity. We recognize that this is frequently, but not always, attributable to a defect in the VWF gene (VWF). Our emphasis remains on practical guidance rather than taxonomic purity. When patients present with mucocutaneous bleeding symptoms suggestive of a primary haemostatic disorder, a quantitative or qualitative abnormality of VWF is a possible cause or contributory factor. During the initial assessment it is important to remember that bleeding histories can be subjective and the disease characteristics can take time to evolve; there is also overlap between symptoms suffered by people with VWD and the normal population (Sadler, 2003; Tosetto et al, 2013). Figure 1 shows the bleeding symptoms in an international study of type 1 VWD patients in comparison with unaffected family members (Tosetto et al, 2006). To improve decisions regarding the significance of bleeding symptoms, attempts have been made to develop a standardized bleeding assessment tool (BAT), with the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH/SSC) BAT being the most recent iteration (Rodeghiero et al, 2010; Rydz & James, 2012). These tools can help predict the likelihood of a bleeding disorder being present and have good negative predictive value but studies evaluating their ability to predict future bleeding episodes are lacking (Tosetto et al, 2013). The plasma level of VWF in normal individuals varies over a six-fold range, from 0·40 to 2·40 iu/ml (Abildgaard et al, 1980), and VWF levels are approximately 25% lower in blood group O individuals than in non-O (Gill et al, 1987). A VWF activity <0·30 iu/ml is usually associated with bleeding symptoms and is more likely to be associated with a mutation in VWF, however these associations are less strong for VWF levels between 0·30 and 0·50 iu/ml (Eikenboom et al, 2006; James et al, 2006). In patients recruited to the MCMDM-1 VWD study, VWF antigen (VWF:Ag) or VWF ristocetin cofactor activity (VWF:RCo) values below 0·40 iu/ml significantly increased the likelihood of type 1 VWD (Tosetto et al, 2006). Amongst 117 obligate carriers of type 3 VWD (type 3 OC) with VWF <0·5 iu/ml, only 26% had bleeding symptoms (Sadler, 2003) and a more recent study (Castaman et al, 2006) did not demonstrate an independent correlation between bleeding score and VWF:Ag in 70 type 3 OC. These patients are also likely to have a normal physiological rise in VWF in response to stress. Therefore, mildly reduced VWF activity in isolation may be insufficient to result in significant bleeding. Nonetheless, some patients with only mildly reduced VWF levels do have significant bleeding symptoms: this is likely to reflect interaction with additional abnormalities in the haemostatic pathway, including mild platelet defects (Millar et al, 2008a; Daly et al, 2009). A study of 280 patients with hereditary mucocutaneous bleeding found abnormalities of VWF and/or platelets in approximately one-third, whilst the majority had no identifiable laboratory abnormality. (Quiroga et al, 2007). Therefore, while identification of laboratory abnormalities may guide management, the primary diagnosis remains ‘abnormal bleeding’; for which risk factors may or may not be identified. Because VWF levels are relatively easy to measure (in comparison to platelet function) VWD has often been diagnosed in patients with bleeding symptoms and VWF levels that are only slightly reduced (0·3–0·5 iu/ml), giving the potentially misleading impression that this is the sole responsible factor. A Bayesian approach to diagnosis of VWD combining laboratory data, personal bleeding history and family data has been evaluated: however an area of uncertainty remained (Tosetto et al, 2008). Thus, caution should be exercised in diagnosing VWD in patients with borderline VWF levels in the range 0·3–0·5 iu/ml in order to avoid the burden of an unnecessary diagnosis and the hazard of failing to complete further necessary investigations. The simplified classification of VWD proposed by the ISTH (Sadler, 1994) is still in common use. Despite the potential for reclassification based on molecular defects, there has been a reluctance to move to a more complex taxonomy; only minor qualifications were introduced when last reviewed (Sadler et al, 2006). Table 1 summarizes how the classification is currently applied. In contrast to type 1 VWD, type 2 variants are usually linked to VWF and usually have a predictable laboratory and clinical phenotype. Misclassification remains an issue, typically between type 1 and type 2M (Nitu-Whalley et al, 2000). In the MCMDM-1 VWD study a third of the families recruited with an historical diagnosis of type 1 VWD had minor multimer abnormalities and a proportion of these were subsequently classified as type 2 VWD (Goodeve et al, 2007; Budde et al, 2008). However the relevance of subtle abnormalities in VWF multimer patterns remains controversial and they lack clear clinical significance (Budde et al, 2008; Castaman et al, 2008; James & Lillicrap, 2012). Classification remains an artificial exercise and although it is of great use for study and research, it does not completely define or predict response to therapy and also has a variable relationship to genetics. From a clinical perspective, any further reclassification of VWD subtypes should focus on clinical utility and, ideally, correlate with responses to therapy. von Willebrand disease cannot be excluded by a normal activated partial thromboplastin time (APTT). Although the overall sensitivity of the Platelet Function Analyser (PFA) for detecting VWD is reported to be 90%, it is close to 100% in type 2 (excluding 2N) and type 3 VWD but lower in type 1 VWD and may be normal when VWF activity is 0·3–0·5 iu/ml. Factor VIII (FVIII), VWF:Ag and measurements of VWF activity are the initial laboratory tests required to make a diagnosis of VWD and must be performed if VWD is suspected. A diagnostic algorithm is shown in Fig 2. Factors that can affect VWF levels (such as anxiety and needle phobia, the combined contraceptive pill, pregnancy and strenuous exercise) were discussed fully in our previous guideline (Laffan et al, 2004). Misdiagnosis can be minimized by ensuring at least two concordant sets of results are obtained. Samples should not be put on ice (Bohm et al, 2006). Factor VIII is measured using an APTT-based one stage clotting assay or chromogenic assay. Although FVIII half-life is regulated by VWF and is frequently reduced in VWD, FVIII levels may be normal in VWD. Plasma VWF:Ag levels are measured by immunological methods, usually by enzyme-linked immunosorbent assay (ELISA) or by automated immunoturbidimetric methods relying on latex particle agglutination. The latter may give falsely high results in the presence of rheumatoid factor. Assessment of the ability of VWF to bind FVIII is discussed below. Here we examine the ability of VWF to support platelet adhesion by binding to platelet glycoprotein Ib (GPIb) and collagen. Binding of VWF to platelet GPIb has traditionally been assessed by ristocetin cofactor activity (VWF:RCo). Ristocetin dimers bind to VWF and induce a conformational change facilitating VWF binding to platelet GPIb and thus cross-linking of platelets. Putative ristocetin binding sites flank the A1 domain, which contains the GPIb binding region. In the traditional assay the agglutination of normal fixed platelets is measured in dilutions of test plasma containing an excess of ristocetin and the patient's VWF:RCo is determined by reference to a plasma standard. The agglutination is dependent on the presence of high molecular weight (HMW) multimers and an intact GPIb binding site. The platelet agglutination method has been automated, with improvement in sensitivity and reproducibility (Lawrie et al, 2011). Other approaches to automation have used a monoclonal antibody to link recombinant GPIb to latex beads (Lawrie et al, 2011, 2013) or magnetic particles (Cabrera et al, 2013). The use of a recombinant GPIb with gain-of-function mutations can remove the requirement for ristocetin (Flood et al, 2011; Lawrie et al, 2013). It must be recognized that using ristocetin rather than shear to induce VWF binding to GPIb is unphysiological. Thus sequence variations affecting the ristocetin binding sites on VWF can result in low VWF:RCo estimation in the absence of a physiological defect of VWF (Flood et al, 2009, 2010). Assays based on monoclonal antibodies directed against the VWF GPIb-binding site, sometimes called ‘VWF activity assays’ have previously failed to detect type 2 VWD (Preston, 1998) and although more recent versions have improved precision and sensitivity, they are not yet sufficiently reliable to replace VWF:RCo (Chen et al, 2011; Favaloro et al, 2012). Pending further data we recommend they are not relied on for diagnosis. The primary collagen binding site of VWF is in the A3 domain. ELISAs are available to measure binding of patient VWF to immobilized collagen. VWF:CB is dependent on the presence of HMW multimers and an intact collagen binding site: assays using type III and type I/III mixtures of collagen have been found to give the best sensitivity to these factors (Favaloro, 2000). Although VWF:RCo and VWF:CB assess different aspects of VWF function, both are sensitive to the loss of HMW multimers and both the VWF:RCo to VWF:Ag ratio and the VWF:CB to VWF:Ag ratio will be reduced in type 2A disease, though the latter ratio is better at differentiating type 2A from type 1 disease (Favaloro et al, 2000). 2A and 2M are qualitative in which VWF function is significantly more reduced than the and the are important in their from type In the MCMDM-1 VWD study the ratio for was with a range of (Goodeve et al, 2007). In a comparison of assays in the lower of normal for was for of collagen (Flood et al, and for Thus patients with a ratio should be to have type 2 VWD and further tests should be performed for Plasma VWF multimer can be using a of different have been for VWF multimer & et al, 2010; et al, 2010). The can be in order to examine the presence of HMW VWF multimers or abnormalities of VWF Von Willebrand factor multimer should not be performed a diagnosis of VWD has been is essential for the classification of type 2 VWD, but if not can be from the ratio (Favaloro, 2007). In normal low of ristocetin are not to platelet agglutination. Platelet agglutination at low ristocetin a of the VWF GPIb which is of type VWD and a wide of severity with and in some patients the only present is it is not practical to on it should be performed when the or ratio is reduced or if is in that this may some mild of type VWD et al, 2009). of the FVIII binding site the and of VWF can FVIII present in the or in with a this rise to type VWD in which plasma VWF:RCo and VWF:CB are often normal but FVIII levels are These mutations may also a type 1 a in FVIII The ability of patient VWF to bind normal FVIII can be assessed using an immunosorbent assay et al, but these assays are and genetic a practical The diagnosis of VWD in and is by to the in an and or and the physiological of VWF In a study of normal the VWF level was iu/ml at 1 of to iu/ml at et al, 1987). Thus the diagnosis of VWD should not be of However when is a deficiency or type 2 disease should be Samples from are to the by and if borderline results are they may to be when the is bleeding for use in are available et al, 2011). for VWD classification and thus may VWF to help disease type or risk of disease or to diagnosis. or A and VWD can be challenging to in the absence of a family The assay can between the two but is not of VWF can mutations in patients that have type individuals lacking these mutations should be for sequence in the VWD and patients present with and can be by studies or by genetic VWD mutations have been between by of VWF whilst mutations affect the or of by the of 2 et al, 2011). using genetic is and can help guide therapy. diagnosis is by families with type 3 VWD, where the have one of the can the which should be to be present in and can subsequently be in a using or sequence and gene may be required to the two VWF mutations although mutations are not yet in et al, 2008). patients where does not VWD genetic can be used to and some patients with mutations may present with a with reduced binding in to reduced binding et al, 2010). for some individuals a clear family history of VWD for family members are and genetic may help provide identification of that or When a diagnosis of VWD is made it is to test degree with or a bleeding In this a diagnosis of VWD may be made on the basis of laboratory patients with (0·3–0·5 iu/ml), family may be on bleeding and family to in VWD the and or containing high VWF or containing The and clinical use of to FVIII and VWF levels by have been reviewed and were discussed in the previous of this guideline Pasi et al, 2004). and have a UK for the of VWD. frequently and sometimes which are not should be by to 1 in the should be if is used in or are use has been by and should not be used in patients are likely to have 2008). Because of clinical and the wide in a of should be in patients with type type 2M and VWD do not have a to use. It is also in patients with bleeding and low VWF as a risk factor In type a frequently et al, This has been as a for and although no have been the response is usually and is not for type VWD 2008). of and responses are in Table 2. The of response will on the defect and there are VWF mutations that have been associated with VWF et al, it is important to measure both the response at and the of response at (Millar et al, Castaman et al, 2013). when the VWF or FVIII response is most minor and may be with (Castaman et al, 2011; et al, 2012). to single is relatively in it is to be used in this group of and close of and for at least is as a or remains a therapy for minor bleeding or to the on or as an therapy to or et al, 2004). A of containing VWF are available for therapy in patients response is for the bleeding or et al, 2009). In which to the plasma and should be although currently available have an haemostatic the characteristics of these are the of the VWF, in the and the of FVIII of A ratio close to 1 is it the VWF has normal structure and function, but the of FVIII is and will according to the high no FVIII and if to a patient with type 3 VWD it will be the FVIII level has to normal et al, et al, 2007). of different a VWF:RCo of et al, but significant in specific activity and in their of HMW multimers (Budde et al, 2006; et al, 2009). is dependent of the plasma VWF:RCo activity to but is dependent on a normal level of Factor VIII et al, et al, 2006). Because FVIII is of large of FVIII may result in an high plasma FVIII bleeding in VWD are bleeding and bleeding can also and type 3 patients can develop or bleeding. When any of these are of and can be et al, the patient is and secondary using should be management of should also be including use of the (Laffan et al, 2004). of in patients with VWD may be in with mild of the disease. type 3 and type 2 variants may be to and there is no that will be when plasma have been into the normal In these should be in a where and laboratory support is available and between responsible including using an in patients with type 1 disease or a of 2A and can often be using with or The level of VWF:RCo and FVIII required will with the but is likely to be if iu/ml. The response to should be measured it is well and also when are The response to a of on lower than the but not to further et al, is required when the VWF has a half-life and in patients with type VWD. When is or the response is a should be The of both VWF and FVIII is approximately iu/ml in of VWD the VWF:RCo and FVIII should be to iu/ml with or In the recent studies have shown by and VWF:RCo iu/ml for et al, 2013). FVIII iu/ml for using a has also et al, and was common VWF:RCo available It does not necessary to the bleeding time or time and these not be should be only in with the ability to these measurements with the required remains a therapy. When bleeding normal plasma levels of VWF platelet may be Platelet is also the of in VWD and may be by et al, 2011; 2011). Because most of VWD are relatively mild and patients do not from is patients with type 3 disease with and with VWD in with an risk factor for as In an international reported that type type 2 and type 1 patients were et al, 2006). The most were bleeding bleeding bleeding and et al, 2006). was to bleeding but was less against bleeding A study also reported that was completely in bleeding et al, 2011). were VWF:RCo In these studies a of VWF were but for a may not be are no data to the level of FVIII or VWF but with in appropriate. at is reported to et al, 2010). of VWF and FVIII rise from in the of pregnancy and with two to of the at to to values a et al, et al, 2003; et al, 2007). with VWD VWF activity does not rise to normal levels pregnancy should be in an that can and Haemophilia Centre and A for management of and the should be between the and Haemophilia Centre and with the The in VWF is often to the VWF deficiency in with type 1 VWD, but not in qualitative or VWF deficiency et al, et al, 2006). may the and bleeding in with type VWD et al, 2012). Von Willebrand factor should be at and at levels have been shown to have to and can be as in type 1 when VWF:RCo iu/ml. In type 2 and 3 VWD, of normal is not therapy. Therefore, is not for use in 2 or 3 VWD of VWF activity has been to normal may if VWF are at the time of bleeding may from It is important that are made of this and to medical help should they develop bleeding. may be to patients with of VWF is both in pregnancy and at et al, but should be in should be in of the sensitivity of the to the of is not pregnancy or the a may be in although is to an in the Where a is at risk of significantly reduced plasma VWF activity and should be with to of therapy can be the is at risk of type 3 VWD. Von Willebrand factor have been in patients with type 3 VWD at a of et al, 2013) but not in VWD develop may present with loss of response to VWF sometimes with an associated et al, 1987). to VWF cannot be by tests or assays et al, 2013). VWF and/or of VWF may be the only of an for type 3 VWD patients with recombinant FVIII by at large to FVIII levels iu/ml et al, recombinant activated et al, et al, platelet et al, 2013) and with may still to of but responses may be A of in a with type 3 VWD and has been et al, 2012). von Willebrand is the used to an loss of VWF It can a wide range of and should be when a patient's symptoms or laboratory results do not their clinical The will to of the cause where this is as of or of are not but associated with frequently to et al, Other VWF plasma and et al, 2011). for patients diagnostic for VWD. with classification of type of patients have had test of of test that a of at of patients with mild have had possible VWD the and in these guidelines is to be and at the time of to the the the British Society for Haematology the any for the of these guidelines. is by a is the for the and of VWD study and support from the an in and the guideline in a of and the has from and advisory board from and and support from and has and support from and advisory board from has on the for and on the advisory of and and from and has no of to this has from and VWF mutation support from has and from and and on advisory for and has on advisory for and has and/or from and

Intronic expansion of the GGGGCC hexanucleotide repeat within the C9ORF72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis/motor neuron disease in both familial and sporadic cases. Initial reports indicate that this variant within the frontotemporal dementia/amyotrophic lateral sclerosis spectrum is associated with transactive response DNA binding protein (TDP-43) proteinopathy. The amyotrophic lateral sclerosis/motor neuron disease phenotype is not yet well characterized. We report the clinical and pathological phenotypes associated with pathogenic C9ORF72 mutations in a cohort of 563 cases from Northern England, including 63 with a family history of amyotrophic lateral sclerosis. One hundred and fifty-eight cases from the cohort (21 familial, 137 sporadic) were post-mortem brain and spinal cord donors. We screened DNA for the C9ORF72 mutation, reviewed clinical case histories and undertook pathological evaluation of brain and spinal cord. Control DNA samples (n = 361) from the same population were also screened. The C9ORF72 intronic expansion was present in 62 cases [11% of the cohort; 27/63 (43%) familial, 35/500 (7%) cases with sporadic amyotrophic lateral sclerosis/motor neuron disease]. Disease duration was significantly shorter in cases with C9ORF72-related amyotrophic lateral sclerosis (30.5 months) compared with non-C9ORF72 amyotrophic lateral sclerosis/motor neuron disease (36.3 months, P < 0.05). C9ORF72 cases included both limb and bulbar onset disease and all cases showed combined upper and lower motor neuron degeneration (amyotrophic lateral sclerosis). Thus, clinically, C9ORF72 cases show the features of a relatively rapidly progressive, but otherwise typical, variant of amyotrophic lateral sclerosis associated with both familial and sporadic presentations. Dementia was present in the patient or a close family member in 22/62 cases with C9ORF72 mutation (35%) based on diagnoses established from retrospective clinical case note review that may underestimate significant cognitive changes in late disease. All the C9ORF72 mutation cases showed classical amyotrophic lateral sclerosis pathology with TDP-43 inclusions in spinal motor neurons. Neuronal cytoplasmic inclusions and glial inclusions positive for p62 immunostaining in non-motor regions were strongly over-represented in the C9ORF72 cases. Extra-motor pathology in the frontal cortex (P < 0.0005) and the hippocampal CA4 subfield neurons (P < 0.0005) discriminated C9ORF72 cases strongly from the rest of the cohort. Inclusions in CA4 neurons were not present in non-C9ORF72 cases, indicating that this pathology predicts mutation status.

Somatic mutations in the phosphatidylinositol/AKT/mTOR pathway cause segmental overgrowth disorders. Diagnostic descriptors associated with PIK3CA mutations include fibroadipose overgrowth (FAO), Hemihyperplasia multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevi, Scoliosis/skeletal and spinal (CLOVES) syndrome, macrodactyly, and the megalencephaly syndrome, Megalencephaly-Capillary malformation (MCAP) syndrome. We set out to refine the understanding of the clinical spectrum and natural history of these phenotypes, and now describe 35 patients with segmental overgrowth and somatic PIK3CA mutations. The phenotypic data show that these previously described disease entities have considerable overlap, and represent a spectrum. While this spectrum overlaps with Proteus syndrome (sporadic, mosaic, and progressive) it can be distinguished by the absence of cerebriform connective tissue nevi and a distinct natural history. Vascular malformations were found in 15/35 (43%) and epidermal nevi in 4/35 (11%) patients, lower than in Proteus syndrome. Unlike Proteus syndrome, 31/35 (89%) patients with PIK3CA mutations had congenital overgrowth, and in 35/35 patients this was asymmetric and disproportionate. Overgrowth was mild with little postnatal progression in most, while in others it was severe and progressive requiring multiple surgeries. Novel findings include: adipose dysregulation present in all patients, unilateral overgrowth that is predominantly left-sided, overgrowth that affects the lower extremities more than the upper extremities and progresses in a distal to proximal pattern, and in the most severely affected patients is associated with marked paucity of adipose tissue in unaffected areas. While the current data are consistent with some genotype-phenotype correlation, this cannot yet be confirmed.

Sudden arrhythmic death syndrome (SADS) describes a sudden death with negative autopsy and toxicological analysis. Cardiac genetic disease is a likely etiology. This study investigated the clinical utility and combined yield of post-mortem genetic testing (molecular autopsy) in cases of SADS and comprehensive clinical evaluation of surviving relatives. We evaluated 302 expertly validated SADS cases with suitable DNA (median age: 24 years; 65% males) who underwent next-generation sequencing using an extended panel of 77 primary electrical disorder and cardiomyopathy genes. Pathogenic and likely pathogenic variants were classified using American College of Medical Genetics (ACMG) consensus guidelines. The yield of combined molecular autopsy and clinical evaluation in 82 surviving families was evaluated. A gene-level rare variant association analysis was conducted in SADS cases versus controls. A clinically actionable pathogenic or likely pathogenic variant was identified in 40 of 302 cases (13%). The main etiologies established were catecholaminergic polymorphic ventricular tachycardia and long QT syndrome (17 [6%] and 11 [4%], respectively). Gene-based rare variants association analysis showed enrichment of rare predicted deleterious variants in RYR2 (p = 5 × 10-5). Combining molecular autopsy with clinical evaluation in surviving families increased diagnostic yield from 26% to 39%. Molecular autopsy for electrical disorder and cardiomyopathy genes, using ACMG guidelines for variant classification, identified a modest but realistic yield in SADS. Our data highlighted the predominant role of catecholaminergic polymorphic ventricular tachycardia and long QT syndrome, especially the RYR2 gene, as well as the minimal yield from other genes. Furthermore, we showed the enhanced utility of combined clinical and genetic evaluation.

Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also because they may undergo progressive adaptation upon long-term culture in vitro. Such adaptation may reflect selection of variants with altered propensity for survival and retention of an undifferentiated phenotype. Elucidating the mechanisms involved will be important for understanding normal self-renewal and commitment to differentiation and for validating the safety of HESC-based therapy. We have investigated this process of adaptation at the cellular and molecular levels through a comparison of early passage (normal) and late passage (adapted) sublines of a single HESC line, H7. To account for spontaneous differentiation that occurs in HESC cultures, we sorted cells for SSEA3, which marks undifferentiated HESC. We show that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes. On the basis of growth potential, ability to re-initiate ES cultures and global transcription profiles, we propose a cellular differentiation hierarchy for maintenance cultures of HESC: normal SSEA3+ cells represent pluripotent stem cells. Normal SSEA3- cells have exited this compartment, but retain multilineage differentiation potential. However, adapted SSEA3+ and SSEA3- cells co-segregate within the stem cell territory, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. As this balance is also an essential feature of cancer, the mechanisms of culture adaptation may mirror those of oncogenesis and tumour progression.

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Whole slide imaging is being used increasingly in research applications and in frozen section, consultation and external quality assurance practice. Digital pathology, when integrated with other digital tools such as barcoding, specimen tracking and digital dictation, can be integrated into the histopathology workflow, from specimen accession to report sign-out. These elements can bring about improvements in the safety, quality and efficiency of a histopathology department. The present paper reviews the evidence for these benefits. We then discuss the challenges of implementing a fully digital pathology workflow, including the regulatory environment, validation of whole slide imaging and the evidence for the design of a digital pathology workstation.

Pyridoxine-dependent epilepsy was recently shown to be due to mutations in the ALDH7A1 gene, which encodes antiquitin, an enzyme that catalyses the nicotinamide adenine dinucleotide-dependent dehydrogenation of l-alpha-aminoadipic semialdehyde/L-Delta1-piperideine 6-carboxylate. However, whilst this is a highly treatable disorder, there is general uncertainty about when to consider this diagnosis and how to test for it. This study aimed to evaluate the use of measurement of urine L-alpha-aminoadipic semialdehyde/creatinine ratio and mutation analysis of ALDH7A1 (antiquitin) in investigation of patients with suspected or clinically proven pyridoxine-dependent epilepsy and to characterize further the phenotypic spectrum of antiquitin deficiency. Urinary L-alpha-aminoadipic semialdehyde concentration was determined by liquid chromatography tandem mass spectrometry. When this was above the normal range, DNA sequencing of the ALDH7A1 gene was performed. Clinicians were asked to complete questionnaires on clinical, biochemical, magnetic resonance imaging and electroencephalography features of patients. The clinical spectrum of antiquitin deficiency extended from ventriculomegaly detected on foetal ultrasound, through abnormal foetal movements and a multisystem neonatal disorder, to the onset of seizures and autistic features after the first year of life. Our relatively large series suggested that clinical diagnosis of pyridoxine dependent epilepsy can be challenging because: (i) there may be some response to antiepileptic drugs; (ii) in infants with multisystem pathology, the response to pyridoxine may not be instant and obvious; and (iii) structural brain abnormalities may co-exist and be considered sufficient cause of epilepsy, whereas the fits may be a consequence of antiquitin deficiency and are then responsive to pyridoxine. These findings support the use of biochemical and DNA tests for antiquitin deficiency and a clinical trial of pyridoxine in infants and children with epilepsy across a broad range of clinical scenarios.

BACKGROUND: BRCA1 and BRCA2 mutations have been associated with prostate cancer (PCa) risk but a wide range of risk estimates have been reported that are based on retrospective studies. OBJECTIVE: To estimate relative and absolute PCa risks associated with BRCA1/2 mutations and to assess risk modification by age, family history, and mutation location. DESIGN, SETTING, AND PARTICIPANTS: This was a prospective cohort study of male BRCA1 (n = 376) and BRCA2 carriers (n = 447) identified in clinical genetics centres in the UK and Ireland (median follow-up 5.9 and 5.3 yr, respectively). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Standardised incidence/mortality ratios (SIRs/SMRs) relative to population incidences or mortality rates, absolute risks, and hazard ratios (HRs) were estimated using cohort and survival analysis methods. RESULTS AND LIMITATIONS: Sixteen BRCA1 and 26 BRCA2 carriers were diagnosed with PCa during follow-up. BRCA2 carriers had an SIR of 4.45 (95% confidence interval [CI] 2.99-6.61) and absolute PCa risk of 27% (95% CI 17-41%) and 60% (95% CI 43-78%) by ages 75 and 85 yr, respectively. For BRCA1 carriers, the overall SIR was 2.35 (95% CI 1.43-3.88); the corresponding SIR at age <65 yr was 3.57 (95% CI 1.68-7.58). However, the BRCA1 SIR varied between 0.74 and 2.83 in sensitivity analyses to assess potential screening effects. PCa risk for BRCA2 carriers increased with family history (HR per affected relative 1.68, 95% CI 0.99-2.85). BRCA2 mutations in the region bounded by positions c.2831 and c.6401 were associated with an SIR of 2.46 (95% CI 1.07-5.64) compared to population incidences, corresponding to lower PCa risk (HR 0.37, 95% CI 0.14-0.96) than for mutations outside the region. BRCA2 carriers had a stronger association with Gleason score ≥7 (SIR 5.07, 95% CI 3.20-8.02) than Gleason score ≤6 PCa (SIR 3.03, 95% CI 1.24-7.44), and a higher risk of death from PCa (SMR 3.85, 95% CI 1.44-10.3). Limitations include potential screening effects for these known mutation carriers; however, the BRCA2 results were robust to multiple sensitivity analyses. CONCLUSIONS: The results substantiate PCa risk patterns indicated by retrospective analyses for BRCA2 carriers, including further evidence of association with aggressive PCa, and give some support for a weaker association in BRCA1 carriers. PATIENT SUMMARY: In this study we followed unaffected men known to carry mutations in the BRCA1 and BRCA2 genes to investigate whether they are at higher risk of developing prostate cancer compared to the general population. We found that carriers of BRCA2 mutations have a high risk of developing prostate cancer, particularly more aggressive prostate cancer, and that this risk varies by family history of prostate cancer and the location of the mutation within the gene.