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BackgroundCopy number variation (CNV) analysis is an integral component of the study of human genomes in both research and clinical settings. Array-based CNV analysis is the current first-tier approach in clinical cytogenetics. Decreasing costs in high-throughput sequencing and cloud computing have opened doors for the development of sequencing-based CNV analysis pipelines with fast turnaround times. We carry out a systematic and quantitative comparative analysis for several low-coverage whole-genome sequencing (WGS) strategies to detect CNV in the human genome.MethodsWe compared the CNV detection capabilities of WGS strategies (short insert, 3 kb insert mate pair and 5 kb insert mate pair) each at 1×, 3× and 5× coverages relative to each other and to 17 currently used high-density oligonucleotide arrays. For benchmarking, we used a set of gold standard (GS) CNVs generated for the 1000 Genomes Project CEU subject NA12878.ResultsOverall, low-coverage WGS strategies detect drastically more GS CNVs compared with arrays and are accompanied with smaller percentages of CNV calls without validation. Furthermore, we show that WGS (at ≥1× coverage) is able to detect all seven GS deletion CNVs >100 kb in NA12878, whereas only one is detected by most arrays. Lastly, we show that the much larger 15 Mbp Cri du chat deletion can be readily detected with short-insert paired-end WGS at even just 1× coverage.ConclusionsCNV analysis using low-coverage WGS is efficient and outperforms the array-based analysis that is currently used for clinical cytogenetics.

This study explores the thermodynamic and vibrational properties of water in the three-dimensional environment of solvated ions and small molecules using molecular simulations. The spectrum of intermolecular vibrations in liquid solvents provides detailed information on the shape of the local potential energy surface, which in turn determines local thermodynamic properties such as the entropy. Here, we extract this information using a spatially resolved extension of the two-phase thermodynamics method to estimate hydration water entropies based on the local vibrational density of states (3D-2PT). Combined with an analysis of solute–water and water–water interaction energies, this allows us to resolve local contributions to the solvation enthalpy, entropy, and free energy. We use this approach to study effects of ions on their surrounding water hydrogen bond network, its spectrum of intermolecular vibrations, and resulting thermodynamic properties. In the three-dimensional environment of polar and nonpolar functional groups of molecular solutes, we identify distinct hydration water species and classify them by their characteristic vibrational density of states and molecular entropies. In each case, we are able to assign variations in local hydration water entropies to specific changes in the spectrum of intermolecular vibrations. This provides an important link for the thermodynamic interpretation of vibrational spectra that are accessible to far-infrared absorption and Raman spectroscopy experiments. Our analysis provides unique microscopic details regarding the hydration of hydrophobic and hydrophilic functional groups, which enable us to identify interactions and molecular degrees of freedom that determine relevant contributions to the solvation entropy and consequently the free energy.

Aim This paper reports on one review of four rapid reviews undertaken to explore the relationships between oral health and general medical conditions, in order to support teams within Public Health England, health practitioners and policy makers. This review aimed to explore the most contemporary evidence on whether poor oral health and cardiovascular disease occurs in the same individuals or populations, to outline the nature of the relationship between these two health outcomes and to discuss the implication of any findings for health services and future research.Methods The review was undertaken by a group comprising consultant clinicians from medicine and dentistry, trainees, public health and academics. The methodology involved a streamlined rapid review process and synthesis of the data.Results The results identified a number of systematic reviews of low to high quality, which suggests that there is: (1) fairly robust evidence of an increased risk of atherosclerotic vascular disease (ASVD) amongst individuals with chronic periodontitis, independent of other established cardiovascular risk factors; (2) there is some evidence that the incidence of caries and tooth loss is higher in patients with cardiovascular disease; and (3) that orofacial pain can presents as the sole symptom of stroke in some patients. The findings are discussed in relation to implications for service and future research.Conclusion There is high quality evidence to support an association between cardiovascular disease and oral health. This evidence is mainly related to the association between chronic periodontitis and atherosclerotic heart disease, and is independent of confounding factors as drawn from epidemiological observational studies.

Few studies have been conducted to understand post-zygotic accumulation of mutations in cells of the healthy human body. We reprogrammed 32 skin fibroblast cells from families of donors into human induced pluripotent stem cell (hiPSC) lines. The clonal nature of hiPSC lines allows a high-resolution analysis of the genomes of the founder fibroblast cells without being confounded by the artifacts of single-cell whole-genome amplification. We estimate that on average a fibroblast cell in children has 1035 mostly benign mosaic SNVs. On average, 235 SNVs could be directly confirmed in the original fibroblast population by ultradeep sequencing, down to an allele frequency (AF) of 0.1%. More sensitive droplet digital PCR experiments confirmed more SNVs as mosaic with AF as low as 0.01%, suggesting that 1035 mosaic SNVs per fibroblast cell is the true average. Similar analyses in adults revealed no significant increase in the number of SNVs per cell, suggesting that a major fraction of mosaic SNVs in fibroblasts arises during development. Mosaic SNVs were distributed uniformly across the genome and were enriched in a mutational signature previously observed in cancers and in de novo variants and which, we hypothesize, is a hallmark of normal cell proliferation. Finally, AF distribution of mosaic SNVs had distinct narrow peaks, which could be a characteristic of clonal cell selection, clonal expansion, or both. These findings reveal a large degree of somatic mosaicism in healthy human tissues, link de novo and cancer mutations to somatic mosaicism, and couple somatic mosaicism with cell proliferation.

OBJECTIVES:Chronic diarrhea caused by primary bile acid diarrhea (PBAD) is a common condition. We have previously shown PBAD is associated with low fasting serum levels of the ileal hormone, fibroblast growth factor 19 (FGF19). FGF19 is a negative regulator of hepatic bile acid synthesis and is stimulated by farnesoid X receptor agonists, which produce symptomatic improvement in PBAD. We aimed to assess possible causes for low serum FGF19 in patients with PBAD.METHODS:Patients with PBAD, defined by reduced 75Se-labelled homocholic acid taurine (SeHCAT) retention, and idiopathic diarrhea controls had measurements of fasting lipids and fasting/post-prandial FGF19 serum profiles. Specific functional variants in candidate genes were investigated in exploratory studies. In further groups, basal and bile acid-stimulated transcript expression was determined in ileal biopsies and explant cultures by quantitative PCR.RESULTS:FGF19 profiles in PBAD patients included low fasting and meal-stimulated responses, which were both strongly correlated with SeHCAT. A subgroup of 30% of PBAD patients had fasting hypertriglyceridemia and higher FGF19. No clear significant differences were found for any genetic variant but there were borderline associations with FGFR4 and KLB. SeHCAT retention significantly correlated with the basal ileal transcript expression of FGF19 (rs=0.59, P=0.03) and apical sodium-dependent bile acid transporter (ASBT) (rs=0.49, P=0.04), and also with the degree of stimulation by chenodeoxycholic acid at 6 h for transcripts of FGF19 (median 184-fold, rs=0.50, P=0.02) and ileal bile acid binding protein (IBABP) (median 2.2-fold, rs=0.47, P=0.04). Median stimulation of FGF19 was lower in patients with SeHCAT retention <10% (P=0.01).CONCLUSIONS:These studies demonstrate a complex, multifactorial etiology of PBAD, including impairments in ileal FGF19 expression and responsiveness.

Endothelial cells contain cigar-shaped secretory organelles called Weibel-Palade bodies (WPBs) that play a crucial role in both hemostasis and the initiation of inflammation. The major cargo protein of WPBs is von Willebrand factor (VWF). In unstimulated cells, this protein is stored in a highly multimerized state coiled into protein tubules, but after secretagogue stimulation and exocytosis it unfurls, under shear force, as long platelet-binding strings. Small GTPases of the Rab family play a key role in organelle function. Using siRNA depletion in primary endothelial cells, we have identified a role for the WPB-associated Rab27a and its effector MyRIP. Both these proteins are present on only mature WPBs, and this rab/effector complex appears to anchor these WPBs to peripheral actin. Depletion of either the Rab or its effector results in a loss of peripheral WPB localization, and this destabilization is coupled with an increase in both basal and stimulated secretion. The VWF released from Rab27a-depleted cells is less multimerized, and the VWF strings seen under flow are shorter. Our results indicate that this Rab/effector complex controls peripheral distribution and prevents release of incompletely processed WPB content.

Andersen-Tawil Syndrome (ATS) is a rare channelopathy caused by mutations in the KCNJ2 gene that encodes the ubiquitously expressed Kir2.1 potassium channel. We describe key findings in a large UK cohort of 52 patients, pertinent to the diagnosis and management of ATS. We report a new point prevalence of0.105 per 100 000 (increased from 0.08 per 100 000).While ATS has historically been considered a triad of episodic weakness, cardiac arrhythmias and dys- morphic features, we show that there is considerable variability to this phenotype. Pure cardiac or muscle phenotypes may exist. The absence of dysmorphic features does not exclude the diagnosis. Similarly, a normal long exercise test was seen in five patients.Importantly, we identify that the phenotype includes a significant risk of cardiac morbidity and mortality with 13% of our cohort requiring cardiac defibrillator or pacemaker insertion and an additional 23% reporting syncope. Syncope has been recently associated with an increased risk of life threatening arhythmic events in this cohort. Severe fixed myopathy was seen in a quarter of our cohort with 14% requiring a wheelchair or gait aid.We additionally describe novel neurological features and report eight new KCNJ2 variants with in vitro functional data. We provide key clinical insights and management recommendations. We also identify new features which are interesting areas for future research and collaboration.v.vivekanandam@ucl.ac.uk

The trafficking of endocytosed receptors through phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing endosomes is thought to attenuate their signaling. Here, we show that the PtdIns(3)P 5-kinase Fab1/PIKfyve controls trafficking but not silencing of endocytosed receptors. Drosophila fab1 mutants contain undetectable phosphatidylinositol 3,5-bisphosphate levels, show profound increases in cell and organ size, and die at the pupal stage. Mutant larvae contain highly enlarged multivesicular bodies and late endosomes that are inefficiently acidified. Clones of fab1 mutant cells accumulate Wingless and Notch, similarly to cells lacking Hrs, Vps25, and Tsg101, components of the endosomal sorting machinery for ubiquitinated membrane proteins. However, whereas hrs, vps25, and tsg101 mutant cell clones accumulate ubiquitinated cargo, this is not the case with fab1 mutants. Even though endocytic receptor trafficking is impaired in fab1 mutants, Notch, Wingless, and Dpp signaling is unaffected. We conclude that Fab1, despite its importance for endosomal functions, is not required for receptor silencing. This is consistent with the possibility that Fab1 functions at a late stage in endocytic receptor trafficking, at a point when signal termination has occurred.

Dental nurse Jenni Pattnireveals how a cuddly dragon is helping oral health educators achieve magic results for patients in the Midlands

Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] is one of the key intracellular second messengers in cells and mobilizes Ca2+ stores in the ER (endoplasmic reticulum). Ins(1,4,5)P3 has a short half-life within the cell, and is rapidly metabolized through one of two pathways, one of which involves further phosphorylation of the inositol ring: Ins(1,4,5)P3 3-kinase (IP3-3K) phosphorylates Ins(1,4,5)P3, resulting in the formation of inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5)P4]. There are three known isoforms of IP3-3K, designated IP3-3KA, IP3-3KB and IP3-3KC. These have differing N-termini, but highly conserved C-termini harbouring the catalytic domain. The three IP3-3K isoforms have different subcellular locations and the B-kinase is uniquely present in both cytosolic and membrane-bound pools. As it is the N-terminus of the B-kinase that differs most from the A- and C-kinases, we have hypothesized that this portion of the protein may be responsible for membrane localization. Although there are no known membrane-targeting protein motifs within the sequence of IP3-3KB, it is found to be tightly associated with the ER membrane. Here, we show that specific regions of the N-terminus of IP3-3KB are necessary and sufficient for efficient membrane localization of the protein. We also report that, in the presence of Ca2+, the kinase domain of IP3-3KB is cleaved from the membrane-anchoring region by calpain.

Salmonellainvade nonphagocytic cells by eliciting their own internalization; upon contact with the host cell, the bacteria induce membrane ruffles highly localized to the point of contact between the invading bacterium and the host cell [1, 2]. The bacterium is then internalized into an unusual cytosolic organelle, the Salmonella-containing vacuole (SCV). Early endosomal markers (including EEA1) have recently been shown to be associated with the SCV shortly after invasion [3]. EEA1, a protein involved in early endosome fusion, is recruited to early endosomal membranes in part by the interaction between its FYVE finger and phosphatidylinositol 3-phosphate [PtdIns(3)P] [4], a characteristic lipid of early endosomes. This suggests a possible role for PtdIns(3)P during Salmonellainfection. To investigate this, we generated a highly specific probe for PtdIns(3)P that was used to follow invasion of Salmonellain nonphagocytic cells. Here, we show that PtdIns(3)P is present on the membranes of SCVs shortly after invasion and also that it is present on the membrane ruffles produced immediately prior to invasion. We also show that this specific probe cycles on and off the membranes of nascent SCVs even when PtdIns 3-kinase activity is inhibited, demonstrating that invading Salmonellainfluence the composition of the membranes that envelop them during invasion.

SBDN charity ambassadors Jenni Pattniand husband Baz have just returned from Uganda, volunteering with Dentaid. Here, they share their experience

Dental nurse Jenni Pattniand her Ugandan-born husband Bhaskar(Baz) joined Dentaid recently to treat hundreds of people who have no other access to dental care in Uganda. Here, Jenni shares their experience

AimsAudit of care for children with Type 2 Diabetes Mellitus in our serviceMethodsData collected from patient records and results software were audited against current ISPAD and AAP guidelines for investigations and management of children with Type 2 diabetes.Results13 children (3 males) diagnosed within the last 4 years were audited. Guidelines were adhered to in 13 (100%) of them. All of them had the required investigations on diagnosis (HbA1c, dyslipidaemia monitoring, renal function, blood pressure and retinopathy screening) and at annual review. All of them were seen by a multidisciplinary team including a Diabetes Clinician, Specialist nurse, Dietician and a psychologist. 10 (76%) were commenced on Metformin within a month of diagnosis. 3 (23%) were controlled on diet only. 3 (23%) of them also had insulin added as adjunct to improve glucose levels and HbA1C. Areas of improvement include discussions surrounding smoking and alcohol which had only been documented for 1 (7%) of these 13 patients.ConclusionIncidence of type 2 diabetes mellitus is on the rise among children and adolescents. We see a significant cohort in our services likely due to the ethnic groups represented in our population. The Team as well as patients and their carers require regular education and input to maintain good standards of care and prevent future complications. Although this audit found that currently most investigations and management plans are meeting guidelines, there needs to be improvement in targeting specific lifestyle habits around adolescent behaviour. There is a need for reimplementing Community programmes like MEND or other subsidised packages from local authorities to aid in exercise opportunities for weight loss.

IntroductionChronic diarrhoea is a common condition which may result from a number of different disorders, including bile acid diarrhoea. Primary bile acid diarrhoea is found in around 36% of patients with functional diarrhoea or diarrhoea-predominant irritable bowel syndrome, but is often undiagnosed. We hypothesised that a positive diagnosis of bile acid diarrhoea would reduce the need in these patients for subsequent investigations to look for alternative diagnoses.MethodPatients previously recruited to a prospective study of chronic diarrhoea, who had SeHCAT testing and subsequent follow-up at our institution, were identified and their electronic records were reviewed. The numbers of defined investigations undertaken between 3 months and 5 years after SeHCAT were compared.Results90 patients were identified with primary bile acid diarrhoea (SeHCAT <15%, n=36) or with idiopathic diarrhoea (SeHCAT >15%, n=54). Follow-up had been performed on 29 and 39 respectively. There was no difference between these groups in the number of previous investigations or the date of the last contact. In the follow-up period, there was no significant difference in the proportion of these patients who had undergone endoscopic procedures (gastroscopy, colonoscopy, sigmoidoscopy). However there was a higher proportion of patients in the SeHCAT-negative group who had undergone other investigations including imaging, physiological tests and blood tests (p=0.037). Use of cross-sectional imaging was significantly higher in this group (p=0.015) with greater proportions having CT (0.44 vs. 0.10) and MRI (0.26 vs. 0.07). Ultrasound use (0.72 vs. 0.55) and the number of blood tests (median 33 vs. 21 tests per patient) were higher in the SeHCAT-negative group whereas the SeHCAT-positive group attended more clinic appointments (p=0.013).ConclusionA positive diagnosis of bile acid diarrhoea, made by a SeHCAT test, resulted in reduced use of diagnostic investigations over the subsequent five years.Disclosure of InterestJ. Turner: None Declared, S Pattni: None Declared, R Appleby: None Declared, J Walters Conflict with: Intercept, Novartis, Conflict with: GE Healthcare

Anderson Tawil Syndrome (ATS) is a rare channelopathy traditionally characterised by periodic paralysis, cardiac arrhythmias and dysmorphic features. Mutations in the KCJN2 gene have been associated with ATS. Accurate and early diagnosis is important in facilitating treatment of episodic paralysis and preventing potentially life-threatening cardiac events.We aim to fully characterise the phenotype in a carefully stratified cohort including cognitive deficits and cardiac risk.66 patients were identified with KCJN2 mutations. Comprehensive clinical information was available for 18 of these patients. Cardiac symptoms were prominent. Two thirds (11) had daily or very frequent palpitations. 27% (5) reported shortness of breath. Serious cardiac complications occurred in 4 patients (ICD insertion, left ventricular dysfunction/cardiomyopathy). 39% (7) reported pain which has previously not been appreciated as part of ATS. Interestingly, 4 patients had fasciculations. 4 patients had minimal decrement (<48%) on Long Exercise Tests (LET) despite having episodic weakness. Lower limb Muscle MRI was abnormal in 6 patients. Excess sleepiness was reported. Neuropsychometric testing suggests slowed processing speed. While dysmorphic features exist, these can be subtle. Short stature is not ubiquitous. Heterogeneity within families was commonly seen.The phenotypic spectrum of ATS is broader than currently appreciated, and data analysis is ongoing.

The article presents questions and answers related to multiple sclerosis (MS) including the impact of MS on young people, the available treatments for MS and how youth clubs can deal with a member with MS.