Publications by authors named "Giuseppe Gallone"

25 Publications

  • Page 1 of 1

The contribution of X-linked coding variation to severe developmental disorders.

Nat Commun 2021 01 27;12(1):627. Epub 2021 Jan 27.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.

Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders.
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http://dx.doi.org/10.1038/s41467-020-20852-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840967PMC
January 2021

Intelligent non-colorimetric indicators for the perishable supply chain by non-wovens with photo-programmed thermal response.

Nat Commun 2020 11 25;11(1):5991. Epub 2020 Nov 25.

NEST, Istituto Nanoscienze-CNR and Scuola Normale Superiore, Piazza San Silvestro 12, I-56127, Pisa, Italy.

Spoiled perishable products, such as food and drugs exposed to inappropriate temperature, cause million illnesses every year. Risks range from intoxication due to pathogen-contaminated edibles, to suboptimal potency of temperature-sensitive vaccines. High-performance and low-cost indicators are needed, based on conformable materials whose properties change continuously and irreversibly depending on the experienced time-temperature profile. However, these systems can be limited by unclear reading, especially for colour-blind people, and are often difficult to be encoded with a tailored response to detect excess temperature over varying temporal profiles. Here we report on optically-programmed, non-colorimetric indicators based on nano-textured non-wovens encoded by their cross-linking degree. This combination allows a desired time-temperature response to be achieved, to address different perishable products. The devices operate by visual contrast with ambient light, which is explained by backscattering calculations for the complex fibrous material. Optical nanomaterials with photo-encoded thermal properties might establish new design rules for intelligent labels.
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http://dx.doi.org/10.1038/s41467-020-19676-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688929PMC
November 2020

Evidence for 28 genetic disorders discovered by combining healthcare and research data.

Nature 2020 10 14;586(7831):757-762. Epub 2020 Oct 14.

GeneDx, Gaithersburg, MD, USA.

De novo mutations in protein-coding genes are a well-established cause of developmental disorders. However, genes known to be associated with developmental disorders account for only a minority of the observed excess of such de novo mutations. Here, to identify previously undescribed genes associated with developmental disorders, we integrate healthcare and research exome-sequence data from 31,058 parent-offspring trios of individuals with developmental disorders, and develop a simulation-based statistical test to identify gene-specific enrichment of de novo mutations. We identified 285 genes that were significantly associated with developmental disorders, including 28 that had not previously been robustly associated with developmental disorders. Although we detected more genes associated with developmental disorders, much of the excess of de novo mutations in protein-coding genes remains unaccounted for. Modelling suggests that more than 1,000 genes associated with developmental disorders have not yet been described, many of which are likely to be less penetrant than the currently known genes. Research access to clinical diagnostic datasets will be critical for completing the map of genes associated with developmental disorders.
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http://dx.doi.org/10.1038/s41586-020-2832-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116826PMC
October 2020

Electrospun ZnO/Poly(Vinylidene Fluoride-Trifluoroethylene) Scaffolds for Lung Tissue Engineering.

Tissue Eng Part A 2020 Dec 25;26(23-24):1312-1331. Epub 2020 Sep 25.

Department of Civil and Industrial Engineering, University of Pisa, Pisa, Italy.

Due to the morbidity and lethality of pulmonary diseases, new biomaterials and scaffolds are needed to support the regeneration of lung tissues, while ideally providing protective effects against inflammation and microbial aggression. In this study, we investigated the potential of nanocomposites of poly(vinylidene fluoride--trifluoroethylene) [P(VDF-TrFE)] incorporating zinc oxide (ZnO), in the form of electrospun fiber meshes for lung tissue engineering. We focused on their anti-inflammatory, antimicrobial, and mechanoelectrical character according to different fiber mesh textures (i.e., collected at 500 and 4000 rpm) and compositions: (0/100) and (20/80) w/w% ZnO/P(VDF-TrFE), plain and composite, respectively. The scaffolds were characterized in terms of morphological, physicochemical, mechanical, and piezoelectric properties, as well as biological response of A549 alveolar epithelial cells in presence of lung-infecting bacteria. By virtue of ZnO, the composite scaffolds showed a strong anti-inflammatory response in A549 cells, as demonstrated by a significant decrease of interleukin (IL) IL-1α, IL-6, and IL-8 expression in 6 h. In all the scaffold types, but remarkably in the aligned composite ones, transforming growth factor β (TGF-β) and the antimicrobial peptide human β defensin-2 (HBD-2) were significantly increased. The ZnO/P(VDF-TrFE) electrospun fiber meshes hindered the biofilm formation by and and the cell/scaffold constructs were able to impede adhesion and and invasiveness, independent of the scaffold type. The data obtained suggested that the composite scaffolds showed potential for tunable mechanical properties, in the range of alveolar walls and fibers. Finally, we also showed good piezoelectricity, which is a feature found in elastic and collagen fibers, the main extracellular matrix molecules in lungs. The combination of all these properties makes ZnO/P(VDF-TrFE) fiber meshes promising for lung repair and regeneration. Impact statement Airway tissue engineering is still a major challenge and an optimally designed scaffold for this application should fulfill a number of key requirements. To help lung repair and regeneration, this study proposes a nondegradable scaffold, with potential for tuning mechanical properties. This scaffold possesses a strong anti-inflammatory character, and is able to hinder microbial infections, sustain epithelial cell growth, and provide physiological signals, like piezoelectricity. The development of such a device could help the treatment of pulmonary deficiency, including the ones induced by inflammatory phenomena, primary and secondary to pathogen infections.
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http://dx.doi.org/10.1089/ten.TEA.2020.0172DOI Listing
December 2020

Biodegradable Polymeric Micro/Nano-Structures with Intrinsic Antifouling/Antimicrobial Properties: Relevance in Damaged Skin and Other Biomedical Applications.

J Funct Biomater 2020 Aug 19;11(3). Epub 2020 Aug 19.

Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.

Bacterial colonization of implanted biomedical devices is the main cause of healthcare-associated infections, estimated to be 8.8 million per year in Europe. Many infections originate from damaged skin, which lets microorganisms exploit injuries and surgical accesses as passageways to reach the implant site and inner organs. Therefore, an effective treatment of skin damage is highly desirable for the success of many biomaterial-related surgical procedures. Due to gained resistance to antibiotics, new antibacterial treatments are becoming vital to control nosocomial infections arising as surgical and post-surgical complications. Surface coatings can avoid biofouling and bacterial colonization thanks to biomaterial inherent properties (e.g., super hydrophobicity), specifically without using drugs, which may cause bacterial resistance. The focus of this review is to highlight the emerging role of degradable polymeric micro- and nano-structures that show intrinsic antifouling and antimicrobial properties, with a special outlook towards biomedical applications dealing with skin and skin damage. The intrinsic properties owned by the biomaterials encompass three main categories: (1) physical-mechanical, (2) chemical, and (3) electrostatic. Clinical relevance in ear prostheses and breast implants is reported. Collecting and discussing the updated outcomes in this field would help the development of better performing biomaterial-based antimicrobial strategies, which are useful to prevent infections.
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http://dx.doi.org/10.3390/jfb11030060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563177PMC
August 2020

Optical Interrogation of Sympathetic Neuronal Effects on Macroscopic Cardiomyocyte Network Dynamics.

iScience 2020 Jul 1;23(7):101334. Epub 2020 Jul 1.

University of Oxford, Department of Physiology, Anatomy and Genetics, British Heart Foundation Centre of Research Excellence, Parks Road, Oxford OX1 3PT, UK; McGill University, Department of Physiology, McIntyre Medical Sciences Building, Room 1128, 3655 Promenade Sir William Osler, Montréal, QC H3G 1Y6, Canada. Electronic address:

Cardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. We present approaches to study neuron-cardiomyocyte interactions involving optogenetic selective probing and all-optical electrophysiology to measure activity in an automated fashion. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiomyocyte network dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac behavior, and the applicability of optogenetics in mechanistic in vitro studies. As arrhythmias are emergent behaviors that involve the coordinated activity of millions of cells, we image at macroscopic scales to capture complex dynamics. We show that neurons can both decrease and increase wave stability and re-entrant activity in culture depending on their induced activity-a finding that may help us understand the often conflicting results seen in experimental and clinical studies.
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http://dx.doi.org/10.1016/j.isci.2020.101334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363704PMC
July 2020

Targeted RNA sequencing enhances gene expression profiling of ultra-low input samples.

RNA Biol 2020 12 28;17(12):1741-1753. Epub 2020 Jun 28.

Department of Psychiatry, University of Oxford , Oxford, UK.

RNA-seq is the standard method for profiling gene expression in many biological systems. Due to the wide dynamic range and complex nature of the transcriptome, RNA-seq provides an incomplete characterization, especially of lowly expressed genes and transcripts. Targeted RNA sequencing (RNA CaptureSeq) focuses sequencing on genes of interest, providing exquisite sensitivity for transcript detection and quantification. However, uses of CaptureSeq have focused on bulk samples and its performance on very small populations of cells is unknown. Here we show CaptureSeq greatly enhances transcriptomic profiling of target genes in ultra-low-input samples and provides equivalent performance to that on bulk samples. We validate the performance of CaptureSeq using multiple probe sets on samples of iPSC-derived cortical neurons. We demonstrate up to 275-fold enrichment for target genes, the detection of 10% additional genes and a greater than 5-fold increase in identified gene isoforms. Analysis of spike-in controls demonstrated CaptureSeq improved both detection sensitivity and expression quantification. Comparison to the CORTECON database of cerebral cortex development revealed CaptureSeq enhanced the identification of sample differentiation stage. CaptureSeq provides sensitive, reliable and quantitative expression measurements on hundreds-to-thousands of target genes from ultra-low-input samples and has the potential to greatly enhance transcriptomic profiling when samples are limiting.
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http://dx.doi.org/10.1080/15476286.2020.1777768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746246PMC
December 2020

Ranbow: A fast and accurate method for polyploid haplotype reconstruction.

PLoS Comput Biol 2020 05 29;16(5):e1007843. Epub 2020 May 29.

Max Planck Institute for Molecular Genetics, Berlin, Germany.

Reconstructing haplotypes from sequencing data is one of the major challenges in genetics. Haplotypes play a crucial role in many analyses, including genome-wide association studies and population genetics. Haplotype reconstruction becomes more difficult for higher numbers of homologous chromosomes, as it is often the case for polyploid plants. This complexity is compounded further by higher heterozygosity, which denotes the frequent presence of variants between haplotypes. We have designed Ranbow, a new tool for haplotype reconstruction of polyploid genome from short read sequencing data. Ranbow integrates all types of small variants in bi- and multi-allelic sites to reconstruct haplotypes. To evaluate Ranbow and currently available competing methods on real data, we have created and released a real gold standard dataset from sweet potato sequencing data. Our evaluations on real and simulated data clearly show Ranbow's superior performance in terms of accuracy, haplotype length, memory usage, and running time. Specifically, Ranbow is one order of magnitude faster than the next best method. The efficiency and accuracy of Ranbow makes whole genome haplotype reconstruction of complex genome with higher ploidy feasible.
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http://dx.doi.org/10.1371/journal.pcbi.1007843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310859PMC
May 2020

Electric field induced alignment of graphene oxide nanoplatelets in polyethersulfone matrix.

Nanotechnology 2020 Apr 20;31(15):155701. Epub 2019 Dec 20.

Nanotechnology group, Engineering Department, Tarbiat Modares University, Tehran, Iran.

In recent years, in order to obtain improved mechanical, thermal, electrical and barrier/transport properties, aligned carbonaceous nanomaterials/polymer nanocomposite films have been receiving growing attention. Correspondingly, the edge oxidized graphene oxide (EOGO) nanoplatelets alignment influence on the structure of the polyethersulfone (PES) membrane films for potential applications in water treatment field has been investigated. Aligned GO/PES nanocomposite membrane films were prepared by non-solvent phase inversion technique after the starting sol phase was preliminarily exposed to high electric fields (50 kV m). Either AC (100, 1000 Hz) or DC mode electric fields were alternatively employed, and the results from both vertical and horizontal field configurations were investigated for structural and morphological comparison. Both XRD, FTIR-ATR, EIS, SEM, TEM and tensile strength analyses were applied in order to characterize the films. The microscopic analyses results have demonstrated successful GO/PES nanocomposite formation and alignment of GO nanoplatelets with the field direction in the matrix at low to moderate (0.02-0.1% wt) GO loadings where the flakes were dispersed and exfoliated sufficiently. However, at higher loading levels (1 and 2% wt) the nanoplateles were mostly agglomerated and the big flakes consisting of irregular plates could not orient their axis parallel to the electric field at the employed field strengths. The results suggest a more effective role of higher frequencies (1000 Hz versus 100 Hz) electric field for alignment of GO nanoplatelets. Simple tensile tests have also similarly confirmed GO alignment under the electric fields at both low (0.1% wt) and moderately high (0.5% wt) GO contents. The tensile strength improvement of the horizontal field processed PES/GO nanocomposite up to 24% compared to its vertical field processed counterpart could be accounted as a proof of the successful alignment of the nanoplatelets. However, EIS results unveiled that non-solvent phase inversion casting method, in its general form, may not be a suitable method for producing materials with tailored properties, due to its random and uncontrollable pore forming mechanism.
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http://dx.doi.org/10.1088/1361-6528/ab646bDOI Listing
April 2020

Contribution of retrotransposition to developmental disorders.

Nat Commun 2019 10 11;10(1):4630. Epub 2019 Oct 11.

Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, Hinxton, CB10 1SA, UK.

Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient's symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies.
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http://dx.doi.org/10.1038/s41467-019-12520-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789007PMC
October 2019

Exome-wide assessment of the functional impact and pathogenicity of multinucleotide mutations.

Genome Res 2019 07 21;29(7):1047-1056. Epub 2019 Jun 21.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA, United Kingdom.

Approximately 2% of de novo single-nucleotide variants (SNVs) appear as part of clustered mutations that create multinucleotide variants (MNVs). MNVs are an important source of genomic variability as they are more likely to alter an encoded protein than a SNV, which has important implications in disease as well as evolution. Previous studies of MNVs have focused on their mutational origins and have not systematically evaluated their functional impact and contribution to disease. We identified 69,940 MNVs and 91 de novo MNVs in 6688 exome-sequenced parent-offspring trios from the Deciphering Developmental Disorders Study comprising families with severe developmental disorders. We replicated the previously described MNV mutational signatures associated with DNA polymerase zeta, an error-prone translesion polymerase, and the APOBEC family of DNA deaminases. We estimate the simultaneous MNV germline mutation rate to be 1.78 × 10 mutations per base pair per generation. We found that most MNVs within a single codon create a missense change that could not have been created by a SNV. MNV-induced missense changes were, on average, more physicochemically divergent, were more depleted in highly constrained genes (pLI ≥ 0.9), and were under stronger purifying selection compared with SNV-induced missense changes. We found that de novo MNVs were significantly enriched in genes previously associated with developmental disorders in affected children. This shows that MNVs can be more damaging than SNVs even when both induce missense changes, and are an important variant type to consider in relation to human disease.
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http://dx.doi.org/10.1101/gr.239756.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633265PMC
July 2019

Pathogenicity and selective constraint on variation near splice sites.

Genome Res 2019 02 26;29(2):159-170. Epub 2018 Dec 26.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom.

Mutations that perturb normal pre-mRNA splicing are significant contributors to human disease. We used exome sequencing data from 7833 probands with developmental disorders (DDs) and their unaffected parents, as well as more than 60,000 aggregated exomes from the Exome Aggregation Consortium, to investigate selection around the splice sites and quantify the contribution of splicing mutations to DDs. Patterns of purifying selection, a deficit of variants in highly constrained genes in healthy subjects, and excess de novo mutations in patients highlighted particular positions within and around the consensus splice site of greater functional relevance. By using mutational burden analyses in this large cohort of proband-parent trios, we could estimate in an unbiased manner the relative contributions of mutations at canonical dinucleotides (73%) and flanking noncanonical positions (27%), and calculate the positive predictive value of pathogenicity for different classes of mutations. We identified 18 patients with likely diagnostic de novo mutations in dominant DD-associated genes at noncanonical positions in splice sites. We estimate 35%-40% of pathogenic variants in noncanonical splice site positions are missing from public databases.
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http://dx.doi.org/10.1101/gr.238444.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360807PMC
February 2019

Quantifying the contribution of recessive coding variation to developmental disorders.

Science 2018 12 8;362(6419):1161-1164. Epub 2018 Nov 8.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK.

We estimated the genome-wide contribution of recessive coding variation in 6040 families from the Deciphering Developmental Disorders study. The proportion of cases attributable to recessive coding variants was 3.6% in patients of European ancestry, compared with 50% explained by de novo coding mutations. It was higher (31%) in patients with Pakistani ancestry, owing to elevated autozygosity. Half of this recessive burden is attributable to known genes. We identified two genes not previously associated with recessive developmental disorders, and , and functionally validated them with mouse and cellular models. Our results suggest that recessive coding variants account for a small fraction of currently undiagnosed nonconsanguineous individuals, and that the role of noncoding variants, incomplete penetrance, and polygenic mechanisms need further exploration.
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http://dx.doi.org/10.1126/science.aar6731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726470PMC
December 2018

Common genetic variants contribute to risk of rare severe neurodevelopmental disorders.

Nature 2018 10 26;562(7726):268-271. Epub 2018 Sep 26.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.

There are thousands of rare human disorders that are caused by single deleterious, protein-coding genetic variants. However, patients with the same genetic defect can have different clinical presentations, and some individuals who carry known disease-causing variants can appear unaffected. Here, to understand what explains these differences, we study a cohort of 6,987 children assessed by clinical geneticists to have severe neurodevelopmental disorders such as global developmental delay and autism, often in combination with abnormalities of other organ systems. Although the genetic causes of these neurodevelopmental disorders are expected to be almost entirely monogenic, we show that 7.7% of variance in risk is attributable to inherited common genetic variation. We replicated this genome-wide common variant burden by showing, in an independent sample of 728 trios (comprising a child plus both parents) from the same cohort, that this burden is over-transmitted from parents to children with neurodevelopmental disorders. Our common-variant signal is significantly positively correlated with genetic predisposition to lower educational attainment, decreased intelligence and risk of schizophrenia. We found that common-variant risk was not significantly different between individuals with and without a known protein-coding diagnostic variant, which suggests that common-variant risk affects patients both with and without a monogenic diagnosis. In addition, previously published common-variant scores for autism, height, birth weight and intracranial volume were all correlated with these traits within our cohort, which suggests that phenotypic expression in individuals with monogenic disorders is affected by the same variants as in the general population. Our results demonstrate that common genetic variation affects both overall risk and clinical presentation in neurodevelopmental disorders that are typically considered to be monogenic.
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http://dx.doi.org/10.1038/s41586-018-0566-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726472PMC
October 2018

De novo mutations in regulatory elements in neurodevelopmental disorders.

Nature 2018 03 21;555(7698):611-616. Epub 2018 Mar 21.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

We previously estimated that 42% of patients with severe developmental disorders carry pathogenic de novo mutations in coding sequences. The role of de novo mutations in regulatory elements affecting genes associated with developmental disorders, or other genes, has been essentially unexplored. We identified de novo mutations in three classes of putative regulatory elements in almost 8,000 patients with developmental disorders. Here we show that de novo mutations in highly evolutionarily conserved fetal brain-active elements are significantly and specifically enriched in neurodevelopmental disorders. We identified a significant twofold enrichment of recurrently mutated elements. We estimate that, genome-wide, 1-3% of patients without a diagnostic coding variant carry pathogenic de novo mutations in fetal brain-active regulatory elements and that only 0.15% of all possible mutations within highly conserved fetal brain-active elements cause neurodevelopmental disorders with a dominant mechanism. Our findings represent a robust estimate of the contribution of de novo mutations in regulatory elements to this genetically heterogeneous set of disorders, and emphasize the importance of combining functional and evolutionary evidence to identify regulatory causes of genetic disorders.
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http://dx.doi.org/10.1038/nature25983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912909PMC
March 2018

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

Genet Med 2018 10 11;20(10):1216-1223. Epub 2018 Jan 11.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.

Purpose: Given the rapid pace of discovery in rare disease genomics, it is likely that improvements in diagnostic yield can be made by systematically reanalyzing previously generated genomic sequence data in light of new knowledge.

Methods: We tested this hypothesis in the United Kingdom-wide Deciphering Developmental Disorders study, where in 2014 we reported a diagnostic yield of 27% through whole-exome sequencing of 1,133 children with severe developmental disorders and their parents. We reanalyzed existing data using improved variant calling methodologies, novel variant detection algorithms, updated variant annotation, evidence-based filtering strategies, and newly discovered disease-associated genes.

Results: We are now able to diagnose an additional 182 individuals, taking our overall diagnostic yield to 454/1,133 (40%), and another 43 (4%) have a finding of uncertain clinical significance. The majority of these new diagnoses are due to novel developmental disorder-associated genes discovered since our original publication.

Conclusion: This study highlights the importance of coupling large-scale research with clinical practice, and of discussing the possibility of iterative reanalysis and recontact with patients and health professionals at an early stage. We estimate that implementing parent-offspring whole-exome sequencing as a first-line diagnostic test for developmental disorders would diagnose >50% of patients.
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http://dx.doi.org/10.1038/gim.2017.246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912505PMC
October 2018

Experimental investigation on the mechanical behavior of polyurethane PICCs after long-term conservation in in vivo-like conditions.

J Vasc Access 2017 Nov 24;18(6):522-529. Epub 2017 Aug 24.

Pain Therapy Unit, S. Chiara Hospital, Pisa - Italy.

Introduction: In a previous paper, the authors investigated the mechanical behavior of several commercial polyurethane peripherally inserted central venous catheters (PICCs) in their 'brand new' condition. The present study represents a second step of the research activity and aims to investigate possible modifications of the PICC mechanical response, induced by long-term conservation in in vivo-like conditions, particularly when used to introduce oncologic drugs.

Methods: Eight 5 Fr single-lumen catheters from as many different vendors, were examined. Several specimens were cut from each of them and kept in a bath at 37°C for 1, 2, 3 and 6 months. Two fluids were used to simulate in vivo-like conditions, i.e. ethanol and Ringer-lactate solutions, the first being chosen in order to reproduce a typical chemical environment of oncologic drugs. The test plan included swelling analyses, uniaxial tensile tests and dynamic mechanical thermal analysis (DMTA).

Results And Conclusions: All tested samples were chemically and mechanically stable in the studied conditions, as no significant weight variation was observed even after six months of immersion in ethanol solution. Uniaxial tensile tests confirmed such a response. For each PICC, very similar curves were obtained from samples tested after different immersion durations in the two fluid solutions, particularly for strains lower than 10%.
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http://dx.doi.org/10.5301/jva.5000772DOI Listing
November 2017

Identification of genetic variants affecting vitamin D receptor binding and associations with autoimmune disease.

Hum Mol Genet 2017 06;26(11):2164-2176

Wellcome Trust Centre for Human Genetics, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7BN, UK.

Large numbers of statistically significant associations between sentinel SNPs and case-control status have been replicated by genome-wide association studies. Nevertheless, few underlying molecular mechanisms of complex disease are currently known. We investigated whether variation in binding of a transcription factor, the vitamin D receptor (VDR), whose activating ligand vitamin D has been proposed as a modifiable factor in multiple disorders, could explain any of these associations. VDR modifies gene expression by binding DNA as a heterodimer with the Retinoid X receptor (RXR). We identified 43,332 genetic variants significantly associated with altered VDR binding affinity (VDR-BVs) using a high-resolution (ChIP-exo) genome-wide analysis of 27 HapMap lymphoblastoid cell lines. VDR-BVs are enriched in consensus RXR::VDR binding motifs, yet most fell outside of these motifs, implying that genetic variation often affects the binding affinity only indirectly. Finally, we compared 341 VDR-BVs replicating by position in multiple individuals against background sets of variants lying within VDR-binding regions that had been matched in allele frequency and were independent with respect to linkage disequilibrium. In this stringent test, these replicated VDR-BVs were significantly (q < 0.1) and substantially (>2-fold) enriched in genomic intervals associated with autoimmune and other diseases, including inflammatory bowel disease, Crohn's disease and rheumatoid arthritis. The approach's validity is underscored by RXR::VDR motif sequence being predictive of binding strength and being evolutionarily constrained. Our findings are consistent with altered RXR::VDR binding contributing to immunity-related diseases. Replicated VDR-BVs associated with these disorders could represent causal disease risk alleles whose effect may be modifiable by vitamin D levels.
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http://dx.doi.org/10.1093/hmg/ddx092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886188PMC
June 2017

Most brain disease-associated and eQTL haplotypes are not located within transcription factor DNase-seq footprints in brain.

Hum Mol Genet 2017 01;26(1):79-89

MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics.

Dense genotyping approaches have revealed much about the genetic architecture both of gene expression and disease susceptibility. However, assigning causality to genetic variants associated with a transcriptomic or phenotypic trait presents a far greater challenge. The development of epigenomic resources by ENCODE, the Epigenomic Roadmap and others has led to strategies that seek to infer the likely functional variants underlying these genome-wide association signals. It is known, for example, that such variants tend to be located within areas of open chromatin, as detected by techniques such as DNase-seq and FAIRE-seq. We aimed to assess what proportion of variants associated with phenotypic or transcriptomic traits in the human brain are located within transcription factor binding sites. The bioinformatic tools, Wellington and HINT, were used to infer transcription factor footprints from existing DNase-seq data derived from central nervous system tissues with high spatial resolution. This dataset was then employed to assess the likely contribution of altered transcription factor binding to both expression quantitative trait loci (eQTL) and genome-wide association study (GWAS) signals. Surprisingly, we show that most haplotypes associated with GWAS or eQTL phenotypes are located outside of DNase-seq footprints. This could imply that DNase-seq footprinting is too insensitive an approach to identify a large proportion of true transcription factor binding sites. Importantly, this suggests that prioritising variants for genome engineering studies to establish causality will continue to be frustrated by an inability of footprinting to identify the causative variant within a haplotype.
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http://dx.doi.org/10.1093/hmg/ddw369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351933PMC
January 2017

Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells.

Nat Immunol 2016 10 22;17(10):1206-1215. Epub 2016 Aug 22.

Department of Biomedicine, University Children's Hospital and University of Basel, Basel, Switzerland.

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.
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http://dx.doi.org/10.1038/ni.3537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033077PMC
October 2016

Cardiac Rhythm Monitoring After Acute Decompensation for Heart Failure: Results from the CARRYING ON for HF Pilot Study.

JMIR Res Protoc 2016 Apr 26;5(2):e62. Epub 2016 Apr 26.

Dept Molecular Medicine, University of Pavia, Italy, Sesto San Giovanni, Milano,, Italy.

Background: There's scarce evidence about cardiovascular events (CV) in patients with hospitalization for acute heart failure (HF) and no indication for immediate device implant.

Objective: The CARdiac RhYthm monitorING after acute decompensatiON for Heart Failure study was designed to assess the incidence of prespecified clinical and arrhythmic events in this patient population.

Methods: In this pilot study, 18 patients (12 (67%) male; age 72±10; 16 (89%) NYHA II-III), who were hospitalized for HF with low left ventricular ejection fraction (LVEF) (<40%) and no immediate indication for device implant received an implantable loop recorder (ILR) before hospital discharge. Follow-up visits were scheduled at 3 and 6 months, and at every 6 months until study closure; device data were remotely reviewed monthly. CV mortality, unplanned CV hospitalization, and major arrhythmic events during follow-up were analyzed.

Results: During a median follow-up of 593 days, major CV occurred in 13 patients (72%); of those, 7 patients had at least 1 cardiac arrhythmic event, 2 had at least a clinical event (CV hospitalization or CV death), and 4 had both an arrhythmic and a CV event. Six (33%) patients experienced 10 major clinical events, 5 of them (50%) were HF related. During follow-up, 2 (11%) patients died due to a CV cause and 3 (16%) patients received a permanent cardiac device.

Conclusions: After an acute HF hospitalization, patients with LVEF<40% and who are not readily eligible for permanent cardiac device implant have a known high incidence of major CV event. In these patients, ILR allows early detection of major cardiac arrhythmias and the ability to react appropriately in a timely manner.

Trial Registration: ClinicalTrials.gov NCT01216670; https://clinicaltrials.gov/ct2/show/NCT01216670.
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http://dx.doi.org/10.2196/resprot.4380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863131PMC
April 2016

A comparative study on the mechanical behavior of polyurethane PICCs.

J Vasc Access 2016 Mar-Apr;17(2):175-81. Epub 2015 Sep 7.

Department of Civil and Industrial Engineering, University of Pisa, Pisa - Italy.

Purpose: This study describes a comparative analysis of eight commercial polyurethane, single-lumen peripherally inserted central venous catheters (PICCs) from different vendors. The aim was to investigate the mechanical response of the catheters providing objective and quantitative data to support a comparison among them. Such data could help nurses and physicians to select a central venous catheter (CVC) not only on the basis of the expected dwell duration or of the assessment of the vessels at the desired insertion site but also of the chemical and mechanical properties of the CVC and of the projected response of the body to these properties.

Methods: An experimental procedure was defined and tests were performed to assess some main characteristics of the PICC lines, including macro and microgeometric features, chemical and physical properties, and mechanical response. Preliminary measurements were performed to accurately define all geometric characteristics, including length, inner and outer diameters, and any inherent initial curvature of the catheter. Micro-geometric features were investigated using surface roughness analysis, optical microscopy, and scanning electron microscopy. Mechanical properties were studied by means of dynamic mechanical thermal analysis, simple uniaxial tensile tests, and kinking tests.

Results: Results are discussed in order to compare the different PICC lines. In particular, they show that polyurethane catheters can have a different mechanical behavior, which might play a role in the onset of pathologic processes and result in an increased risk and incidence of catheter-related complications.

Conclusions: This study provides useful information that can help identifying and facilitate the choice of a PICC.
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http://dx.doi.org/10.5301/jva.5000452DOI Listing
December 2016

Mutations in ZMYND10, a gene essential for proper axonemal assembly of inner and outer dynein arms in humans and flies, cause primary ciliary dyskinesia.

Am J Hum Genet 2013 Aug 25;93(2):346-56. Epub 2013 Jul 25.

Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK.

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects.
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http://dx.doi.org/10.1016/j.ajhg.2013.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738835PMC
August 2013

Vitamin D receptor ChIP-seq in primary CD4+ cells: relationship to serum 25-hydroxyvitamin D levels and autoimmune disease.

BMC Med 2013 Jul 12;11:163. Epub 2013 Jul 12.

Medical Research Council Functional Genomics Unit and Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, UK.

Background: Vitamin D insufficiency has been implicated in autoimmunity. ChIP-seq experiments using immune cell lines have shown that vitamin D receptor (VDR) binding sites are enriched near regions of the genome associated with autoimmune diseases. We aimed to investigate VDR binding in primary CD4+ cells from healthy volunteers.

Methods: We extracted CD4+ cells from nine healthy volunteers. Each sample underwent VDR ChIP-seq. Our results were analyzed in relation to published ChIP-seq and RNA-seq data in the Genomic HyperBrowser. We used MEMEChIP for de novo motif discovery. 25-Hydroxyvitamin D levels were measured using liquid chromatography-tandem mass spectrometry and samples were divided into vitamin D sufficient (25(OH)D ≥75 nmol/L) and insufficient/deficient (25(OH)D <75 nmol/L) groups.

Results: We found that the amount of VDR binding is correlated with the serum level of 25-hydroxyvitamin D (r = 0.92, P= 0.0005). In vivo VDR binding sites are enriched for autoimmune disease associated loci, especially when 25-hydroxyvitamin D levels (25(OH)D) were sufficient (25(OH)D ≥75: 3.13-fold, P<0.0001; 25(OH)D <75: 2.76-fold, P<0.0001; 25(OH)D ≥75 enrichment versus 25(OH)D <75 enrichment: P= 0.0002). VDR binding was also enriched near genes associated specifically with T-regulatory and T-helper cells in the 25(OH)D ≥75 group. MEME ChIP did not identify any VDR-like motifs underlying our VDR ChIP-seq peaks.

Conclusion: Our results show a direct correlation between in vivo 25-hydroxyvitamin D levels and the number of VDR binding sites, although our sample size is relatively small. Our study further implicates VDR binding as important in gene-environment interactions underlying the development of autoimmunity and provides a biological rationale for 25-hydroxyvitamin D sufficiency being based at 75 nmol/L. Our results also suggest that VDR binding in response to physiological levels of vitamin D occurs predominantly in a VDR motif-independent manner.
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http://dx.doi.org/10.1186/1741-7015-11-163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710212PMC
July 2013

Bio::Homology::InterologWalk--a Perl module to build putative protein-protein interaction networks through interolog mapping.

BMC Bioinformatics 2011 Jul 18;12:289. Epub 2011 Jul 18.

Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.

Background: Protein-protein interaction (PPI) data are widely used to generate network models that aim to describe the relationships between proteins in biological systems. The fidelity and completeness of such networks is primarily limited by the paucity of protein interaction information and by the restriction of most of these data to just a few widely studied experimental organisms. In order to extend the utility of existing PPIs, computational methods can be used that exploit functional conservation between orthologous proteins across taxa to predict putative PPIs or 'interologs'. To date most interolog prediction efforts have been restricted to specific biological domains with fixed underlying data sources and there are no software tools available that provide a generalised framework for 'on-the-fly' interolog prediction.

Results: We introduce Bio::Homology::InterologWalk, a Perl module to retrieve, prioritise and visualise putative protein-protein interactions through an orthology-walk method. The module uses orthology and experimental interaction data to generate putative PPIs and optionally collates meta-data into an Interaction Prioritisation Index that can be used to help prioritise interologs for further analysis. We show the application of our interolog prediction method to the genomic interactome of the fruit fly, Drosophila melanogaster. We analyse the resulting interaction networks and show that the method proposes new interactome members and interactions that are candidates for future experimental investigation.

Conclusions: Our interolog prediction tool employs the Ensembl Perl API and PSICQUIC enabled protein interaction data sources to generate up to date interologs 'on-the-fly'. This represents a significant advance on previous methods for interolog prediction as it allows the use of the latest orthology and protein interaction data for all of the genomes in Ensembl. The module outputs simple text files, making it easy to customise the results by post-processing, allowing the putative PPI datasets to be easily integrated into existing analysis workflows. The Bio::Homology::InterologWalk module, sample scripts and full documentation are freely available from the Comprehensive Perl Archive Network (CPAN) under the GNU Public license.
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http://dx.doi.org/10.1186/1471-2105-12-289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161927PMC
July 2011