Center For Human Genetics Research Articles

Multioviz: an interactive platform for in silico perturbation and interrogation of gene regulatory networks

In this paper, we aim to build a platform that will help bridge the gap between high-dimensional computation and wet-lab experimentation by allowing users to interrogate genomic signatures at multiple molecular levels and identify best next actionable steps for downstream decision making. We introduce Multioviz: a publicly accessible R package and web application platform to easily perform in silico hypothesis testing of generated gene regulatory networks. We demonstrate the utility of Multioviz by conducting an end-to-end analysis in a statistical genetics application focused on measuring the effect of in silico perturbations of complex trait architecture. By using a real dataset from the Wellcome Trust Centre for Human Genetics, we both recapitulate previous findings and propose hypotheses about the genes involved in the percentage of immune CD8+ cells found in heterogeneous stocks of mice. Source code for the MultiovizR package is available at https://github.com/lcrawlab/multio-viz and an interactive version of the platform is available at https://multioviz.ccv.brown.edu/.

Endocrine manifestations in adults with 22q11.2 deletion syndrome: a retrospective single-center cohort study

Introduction and objectivePatients with the 22q11.2 deletion syndrome (22q11DS) frequently display cardiological and psychiatric diseases, but are also at increased risk for endocrine manifestations. The aim of this study was to evaluate the screening, prevalence, and management of hypoparathyroidism and thyroid disease in patients with 22q11DS, to evaluate the metabolic profile, and to compare these results with current literature and guidelines.DesignWe performed a retrospective study of patients with genetically confirmed 22q11DS, followed at the center for human genetics of the University Hospitals Leuven, resulting in a cohort of 75 patients. Medical history, medication, and laboratory results concerning hypoparathyroidism, thyroid dysfunction, and the metabolic profile were collected.ResultsOf the total cohort, 26 patients (35%) had at least one hypocalcaemic episode. During hypocalcaemia, parathyroid hormone (PTH) was measured in only 12 patients with 11 having normal or low PTH, confirming a diagnosis of hypoparathyroidism. Recurrent episodes of hypocalcaemia occurred in seventeen patients (23%). Adherence to the guidelines was low, with 13% of patients having a yearly serum calcium evaluation, 12% receiving daily calcium supplements, and 20% receiving non-active vitamin D. Hypothyroidism was present in 31 patients (44%) and hyperthyroidism in 6 patients (8%). Information on body mass index (BMI) was available in 52 patients (69%), of which 38% were obese (BMI ≥ 30 kg/m2).ConclusionHypoparathyroidism, hypothyroidism, and obesity are common endocrine manifestations in patients with 22q11DS but are probably underdiagnosed and undertreated, indicating the need for multidisciplinary follow-up including an endocrinologist.

Joint regression analysis of multiple traits based on genetic relationships.

Polygenic scores (PGSs) are widely available and employed in genomic data analyses for predicting and understanding genetic architectures. Existing approaches either require information on SNP level, do not infer clusters of traits sharing genetic characteristic, or do not have any immediate predictive properties. Here, we present geneJAM, which is a novel clustering and estimation method using PGSs for inferring a genetic relationship among multiple, simultaneously measured and potentially correlated traits in a multivariate GWAS.Using graphical lasso, we estimate a sparse covariance matrix of the PGSs and obtain clusters of traits sharing genetic characteristics. We use the clusters to specify the structure of the error covariance matrix of a generalized least squares (GLS) model and use the feasible GLS estimator for estimating a linear regression model with a certain unknown degree of correlation between the residuals.The method suits many biology studies well with traits embedded in some genetic functioning groups and facilitates development of the PGS research. We compare the method with fully parametric techniques on simulated data and illustrate the utility of the methods by examining a heterogeneous stock mouse data set from the Wellcome Trust Centre for Human Genetics. We demonstrate that the method successfully identifies clusters of traits and increases precision, power, and computational efficiency. GeneJAM is implemented in R and available at: https://github.com/abuchardt/geneJAM.

Leveraging the genetic correlation between traits improves the detection of epistasis in genome-wide association studies.

Epistasis, commonly defined as the interaction between genetic loci, is known to play an important role in the phenotypic variation of complex traits. As a result, many statistical methods have been developed to identify genetic variants that are involved in epistasis, and nearly all of these approaches carry out this task by focusing on analyzing one trait at a time. Previous studies have shown that jointly modeling multiple phenotypes can often dramatically increase statistical power for association mapping. In this study, we present the "multivariate MArginal ePIstasis Test" (mvMAPIT)-a multioutcome generalization of a recently proposed epistatic detection method which seeks to detect marginal epistasis or the combined pairwise interaction effects between a given variant and all other variants. By searching for marginal epistatic effects, one can identify genetic variants that are involved in epistasis without the need to identify the exact partners with which the variants interact-thus, potentially alleviating much of the statistical and computational burden associated with conventional explicit search-based methods. Our proposed mvMAPIT builds upon this strategy by taking advantage of correlation structure between traits to improve the identification of variants involved in epistasis. We formulate mvMAPIT as a multivariate linear mixed model and develop a multitrait variance component estimation algorithm for efficient parameter inference and P-value computation. Together with reasonable model approximations, our proposed approach is scalable to moderately sized genome-wide association studies. With simulations, we illustrate the benefits of mvMAPIT over univariate (or single-trait) epistatic mapping strategies. We also apply mvMAPIT framework to protein sequence data from two broadly neutralizing anti-influenza antibodies and approximately 2,000 heterogeneous stock of mice from the Wellcome Trust Centre for Human Genetics. The mvMAPIT R package can be downloaded at https://github.com/lcrawlab/mvMAPIT.

Neurodevelopmental profile and stages of regression in Phelan-McDermid syndrome.

To characterize the neurodevelopmental profile of patients with Phelan-McDermid syndrome (PMS) and describe the nature and trajectory of regression. This was a retrospective, monocentric study examining the clinical and developmental data of 24 patients (average age=25 years 6 months, range=6-56 years, n=13 males) with a confirmed 22q13.3 terminal deletion carried out at the Centre for Human Genetics, University Hospital Leuven. The neurodevelopmental profile of individuals with PMS was examined, combining both cross-sectional and longitudinal data obtained by systematic review of digital medical records. Remarkable loss of skills was present in 19 individuals affecting both language and motor skills. The first manifestations of neurodevelopmental regression occurred, on average, at the age of 7 years 6 months (range=5-11 years). Language skills (active vocabulary) were primarily affected followed by, in order of loss, psychosocial adaptability, fine motor skills, and walking ability. The course of regression was characterized by a distinctive four-stage pattern. The first stage often occurred around mid-childhood and was defined by a pronounced and abrupt decline of language skills. This stage was generally followed by the second stage where a (prolonged) period of stagnation of regression was seen. The third stage was defined by acute neuropsychiatric decline (e.g. catatonia, hallucinations, psychosis). Acute events such as severe sickness, hormonal shifts, and psychosocial stress frequently preceded the fourth and final stage, which was characterized by severe neuromotor degeneration. Neurodevelopmental regression should be considered as a key feature of PMS. We present a four-stage model of neurodevelopmental regression, entailing language skills, fine and gross motor function, and psychosocial adaptation, which can be applied in future practice and research.

Navigating the uncertainties of next-generation sequencing in the genetics clinic.

This study explores the different manifestations and navigations of uncertainty in the practice of diagnostic next-generation sequencing (NGS) testing. Drawing upon multi-sited fieldwork conducted at a large Centre for Human Genetics in Belgium, we analyse how uncertainty takes shape and is managed in the different steps of the diagnostic process: starting from the testing offer, to the analysis in the lab, the multidisciplinary team meetings (MDTs) and ending with the consultation with the patient. Building on interviews with genetic healthcare professionals and their patients and observations in consultations and MDTs, our empirical work underlines the duality of uncertainty as both burdensome and productive. Building on the existing literature on uncertainty in medicine and NGS, our analysis shows the ontological politics at play in the everyday uncertainty work in this CHG. We show how the, at times, contrasting ways of dealing with uncertainty lead to friction but also result in constructive negotiation and collaboration between actors, making use of multiple types of evidence and expertise. By not only minimising but also sustaining or inviting uncertainty, genetic healthcare professionals are able to advance the practices around NGS in a way that matches their multidisciplinary understandings, considerations and more normative stances.

Value of DNA testing in the diagnosis of sickle-cell anemia in childhood in an environment with a high prevalence of other causes of anemia.

BackgroundSickle‐cell anemia (SCA) is the most common genetic disease worldwide caused by a single mutation in the gene HBB. DNA testing can help to clarify the diagnosis when Hb electrophoresis is inconclusive. We evaluated the usefulness and feasibility of DNA‐based diagnosis of SCA in rural Central Africa.MethodsThis is a cross‐sectional study conducted from November 2016 to end October 2017 in the Hôpital Saint Luc de Kisantu, located 120 km from Kinshasa. This hospital offers the management of SCA patients, mainly identified using the Sickling test (Emmel test) combined with clinical features. We included patients aged 6 months to 18 years locally diagnosed as SCA, and we collected clinical and hematological data. All patients were offered Hb electrophoresis and DNA testing at the Center for Human Genetics of the University of Kinshasa.ResultsThis study included 160 patients. Hemoglobin capillary electrophoresis suggested that 136 (85%) were homozygote SS, 13 (8.1%) were heterozygote (AS), and 11 (6.9%) were homozygote normal (AA). DNA testing confirmed these electrophoresis findings, with the exception of four patients, two AS in electrophoresis were found SS due to recent transfusion, and two SS in electrophoresis were found AS because they have compound heterozygous form S/β°‐thalassemia. The diagnosis of SCA was therefore wrongly ascertained with Emmel test in 15% of patients.ConclusionThis study reveals a high proportion of false‐positive SCA diagnoses in a rural environment in Central Africa. This underlines the importance of DNA testing in conjunction with Hb electrophoresis.

P-561 Long-read amplicon guided haplotype imputation enabling comprehensive preimplantation genetic testing in families with de novo pathogenic variants

Abstract Study question Can we use comprehensive genome-wide haplotyping PGT for couples carrying de novo mutations, given the absence of phasing references to establish the disease-associated haplotype? Summary answer Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. What is known already Preimplantation genetic testing (PGT) aims to select embryos devoid of inherited pathogenic variants. Current comprehensive genome-wide haplotyping PGT methods cannot be applied for couples where one of the partners carries a de novo mutation, as absence of affected close relatives restricts variant phasing to establish the disease-associated haplotype. For such families targeted approaches are currently used instead, thus missing genome-wide embryo analysis. Study design, size, duration To overcome the forementioned limitation, we developed a long-read amplicon guided haplotype imputation method. From January 2017 till June 2021 thirty-two couples were enrolled into the PGT program at the Centre for Human Genetics, UZ Leuven, in the context of a de novo variant causing a known monogenic disorder in one of the two partners. Participants/materials, setting, methods Genomic DNA from the partner carrying the variant (proband) and his/her parents was used for trio analysis via long-range PCR and long-read amplicon sequencing using the PacBio RSII and/or Oxford Nanopore platforms. Targeted haplotype phasing was then performed to impute the disease-associated allele. Main results and the role of chance The parental origin of the mutant allele was identified in 20 patients, resulting in the current 62.5% success rate. Most de novo mutations occurred on the paternal allele. In the remaining families, the region of interest either had no SNPs or had insufficient number of informative SNPs linked to mutation. From the successfully analyzed couples, eight have proceeded to PGT cycle and so far, three disease-free children have been born. Limitations, reasons for caution A costly family-specific work-up is required, allowing nevertheless the implementation of a generic genome-wide method at the embryo analysis stage. The sequencing cost itself is anticipated to decrease in time. To avoid non-informative results, the analysis of multiple amlpicons is recommended. Wider implications of the findings Current practices for the handling of couples with de novo variants are mostly targeted, require the combination of direct and indirect approaches and often require the multiple biopsies. Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. Trial registration number not applicable

Knowledge, attitudes and preferences regarding reproductive genetic carrier screening among reproductive-aged men and women in Flanders (Belgium).

Through carrier screening couples at-risk of conceiving a child with an autosomal recessive or X-linked condition can be identified prior to conception. The aim of this study was to assess knowledge, attitudes and preferences regarding reproductive genetic carrier screening (RGCS) among reproductive-aged men and women in Flanders (Belgium). Women and men of reproductive age visiting their pharmacist were invited to answer a self-administered questionnaire. Prior to filling in the questionnaire, participants were asked to read an information leaflet explaining some key concepts about RGCS. Our sample included 387 individuals of reproductive age, of which 68.5% were female and 31.5% were male. Most of the participants were below 34 years old (72.9%), didn't have children (68.6%) and were currently in a relationship (69.1%). Offering RGCS to couples that want to have children was found acceptable by 86% of participants. However, fewer participants would consider RGCS for themselves in the future (61%). We observed a positive correlation between attitude score/knowledge score and the intention to have RGCS. Half of the participants (50.9%) preferred the disclosure of individual test results. Most of participants indicated that RGCS should be offered through the gynecologist (81.1%), followed by the GP (71.5%) and the Centre for Human Genetics (64.8%). About 68.9% of participants were willing to pay out-of-pocket for an RGCS test. We recommend that RGCS should ideally be implemented through a tailored implementation strategy whereby individual needs and preferences can be taken into account.

Telegenetics: The experience of an Indian center (Centre for Human Genetics) during the COVID-19 pandemic.

Because of the lockdowns and restrictions placed on non‐emergency medical services due to the COVID‐19 pandemic, we were prompted to set up telegenetic services for patients and families with genetic disorders. Genetic medicine poses special challenges because the unit of consultation and counseling is often the family and not just the individual. We describe here our experience over eight months in 2020 in evaluating 539 families with genetic disorders on a virtual platform. Patients from urban and rural districts of Karnataka and neighboring states received telegenetic consultation. Families were phoned by genetic counselors 14–28 days after the initial consultation to measure feedback. One member of each family was invited to complete a modified 9‐item Telehealth Satisfaction Scale (TeSS scale). Of 293 respondents, approximately 87.3% reported satisfaction with the visual and audio quality of online contact and 86.7% on saving travel time and expenses. A shorter waiting time for appointments as compared to in‐person appointments in the previous year was seen in approximately 90%. Nearly 87% reported satisfaction with online genetic consultation; however, 74% of these indicated a preference for a face‐to‐face appointment. The reasons for this included a cultural perception of confidence instilled by meeting medical specialists in person. Telegenetics presents unusual advantages in India because of the high usage of smartphones, unlimited Internet data as a feature of most Internet plans, free web‐based video applications, and digital payments. We suggest that telegenetics may be an alternative in providing a hybrid model of care in non‐emergency situations especially where resources are limited.

Multi-scale inference of genetic trait architecture using biologically annotated neural networks.

In this article, we present Biologically Annotated Neural Networks (BANNs), a nonlinear probabilistic framework for association mapping in genome-wide association (GWA) studies. BANNs are feedforward models with partially connected architectures that are based on biological annotations. This setup yields a fully interpretable neural network where the input layer encodes SNP-level effects, and the hidden layer models the aggregated effects among SNP-sets. We treat the weights and connections of the network as random variables with prior distributions that reflect how genetic effects manifest at different genomic scales. The BANNs software uses variational inference to provide posterior summaries which allow researchers to simultaneously perform (i) mapping with SNPs and (ii) enrichment analyses with SNP-sets on complex traits. Through simulations, we show that our method improves upon state-of-the-art association mapping and enrichment approaches across a wide range of genetic architectures. We then further illustrate the benefits of BANNs by analyzing real GWA data assayed in approximately 2,000 heterogenous stock of mice from the Wellcome Trust Centre for Human Genetics and approximately 7,000 individuals from the Framingham Heart Study. Lastly, using a random subset of individuals of European ancestry from the UK Biobank, we show that BANNs is able to replicate known associations in high and low-density lipoprotein cholesterol content.

­­­The Antigenic Anatomy of SARS-CoV-2 Receptor Binding Domain

Antibodies are crucial to immune protection against SARS-CoV-2 with some in emergency use as therapeutics. Here we identified 377 human monoclonal antibodies (mAbs) recognizing the viral spike, and focused on 80 which bind the receptor binding domain (RBD). By mapping antigenic sites using a unique computational methodology and comparing with inhibitory activity, we show that binding sites are widely dispersed, but neutralizing epitopes highly focused. Nearly all highly potent neutralizing mAbs (IC50< 0.1mg/ml) block receptor interaction, although one binds a unique epitope in the N-terminal domain. Many mAbs use public V-genes and are close to germline, boding well for vaccine responses. 19 Fab-antigen structures, some as RBD complexed with two Fabs, reveal two novel modes of engagement for potently inhibitory mAbs. Several Fabs are glycosylated, enhancing neutralisation for three, for two of which the sugar contacts the antigen. The most potent mAbs protect, prophylactically or therapeutically, in animal models. Ethical Approval: Patients were recruited into the Sepsis Immunomics project [Oxford REC C, reference:19/SC/0296] ISARIC/WHO Clinical Characterisation Protocol for Severe Emerging Infections [Oxford REC C, reference 13/SC/0149]. Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01). Funding: The Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (ID: 2018-I2M-2-002); Medical Research Council, UK. National Institute for Health Research Biomedical Research Centre Funding Scheme (to G.R.S.), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant number: 2018-I2M-2-002) to D.I.S. and G.R.S., and G.R.S. is supported as a Wellcome Trust Senior Investigator (grant 095541/A/11/Z). H.M.E.D. and J.Ren are supported by the Wellcome Trust (101122/Z/13/Z), Y.Z. by Cancer Research UK (C375/A17721) and D.I.S. by the UK Medical Research Council (MR/N00065X/1). D.I.S. and GRS are Jenner Investigators. N.M.K is supported by T32AI007172. The National Institute for Health Research Biomedical Research Centre Funding Scheme supports G.R.S. We are also grateful for a Fast Grant from Fast Grants, Mercatus Center to support the isolation of human monoclonal antibodies to SARS-2 and Schmidt Futures for support of this work. G.R.S. is also supported as a Wellcome Trust Senior Investigator (grant 095541/A/11/Z). This is a contribution from the UK Instruct-ERIC Centre. The Wellcome Centre for Human Genetics is supported by the Wellcome Trust (grant 090532/Z/09/Z). We acknowledge Diamond Light Source for time on Beamline I03 under Proposal lb27009 and for electron microscope time at the UK national electron bio-imaging centre (eBIC), Proposal BI26983. Computational aspects were supported by the Wellcome Trust Core Award Grant Number 203141/Z/16/Z and the NIHR Oxford BRC. This study was supported by grants from NIH (R01 AI157155) and the Defense Advanced Research Project Agency (HR001117S0019). J.B.C. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship. Competing Interest: M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, Carnival Corporation and on the Scientific Advisory Boards of Moderna and Immunome. The M.S.D. laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. GRS sits on the GSK Vaccines Scientific Advisory Board.

Prolonged Infectious SARS-CoV-2 Shedding from an Immunocompromised Patient

Long-term SARS-CoV-2 shedding was observed from the upper respiratory tract of an immunocompromised patient with chronic lymphocytic leukemia and acquired hypogammaglobulinemia. Shedding of SARS-CoV-2 genomic and subgenomic RNA was observed up to 105 days, and infectious virus up to 70 days past the initial diagnosis. The infection was not cleared after a first treatment with convalescent plasma, suggesting limited impact on SARS-CoV-2 in the upper respiratory tract. SARS-CoV-2 RNA was no longer detected several weeks after a second transfusion of convalescent plasma. There was marked within-host genomic evolution of SARS-CoV-2, with continuous turnover of dominant viral variants. However, replication kinetics in VeroE6 cells and primary human alveolar epithelial tissues were not affected. Our data indicate that certain immunocompromised patients may shed infectious virus for longer durations than previously recognized. Detection of of subgenomic RNA is recommended in persistently SARS-CoV-2 positive individuals as a proxy for shedding of infectious virus.Funding: This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID). T.A.B. is supported by the Medical Research Council UK (MR/S007555/1). The Wellcome Centre for Human Genetics is supported by Wellcome Centre grant 203141/Z/16Z. Conflict of Interest: The authors declare no competing interests.

Abstract P6-08-03: Germline mutational landscape in 5422 individuals at risk for hereditary breast and ovarian cancer who underwent multi-gene panel testing

Abstract Background: The introduction of multi-gene panel testing and improved awareness under patients and physicians has led to an increase of individuals with known germline pathogenic variants in hereditary breast and ovarian cancer (HBOC) genes. Significant regional differences exist in germline mutational landscape. We aimed to report the findings from multi-gene panel testing in a large Belgian cohort of individuals at risk for HBOC. Methods: All individuals who underwent multi-gene panel testing for HBOC at the Center for Human Genetics of the University Hospitals Leuven since the introduction of the panel were included (March 2016-April 2019). All included individuals were considered candidates for HBOC-panel testing by the requesting physician based on a personal or familial history of breast and/or ovarian cancer. Testing criteria from the Belgian Society of Human Genetics (www.beshg.be/download/guidelines/Guidelines_HBOC_2018.pdf) were met in the vast majority. The panel used was the BRCA Hereditary Cancer MASTR Plus® (Agilent, Belgium), with sequencing of BARD1, BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, TP53, MRE11A, RAD50, NBN, FAM175A, ATM, PALB2, STK11, MEN1, PTEN, CDH1, MUTYH, CHEK2, BLM, XRCC2, EPCAM, MLH1, MSH6, PMS2 and MSH2. Sequencing was performed by NGS on a Miseq platform (Illumina). Genomic deletions and duplications in BRCA1 and BRCA2 were investigated with multiplex ligation-dependent probe amplification. We hereby report on the frequency of pathogenic and likely pathogenic germline variants in this population. Results: In 5422 individuals who underwent multi-gene panel testing, we detected 665 pathogenic or likely pathogenic variants in 639 patients (11,7%). In 25 patients (0.46%), more than one relevant alteration was detected with double heterozygosity in 24 individuals and triple heterozygosity in one. Germline variants in BRCA1 and BRCA2 were detected in 178 (3.3%) and 144 (2.7%) patients, resulting in a fraction of 26,4% and 21,4% of detected variants respectively. Relevant alterations in CHEK2, ATM, PALB2 and TP53 were observed in 135 (2.5%), 93 (1.7%), 26 (0.5%) and 11 (0.2%) patients respectively, accounting together for 39.3% of detected variants. Alterations in BRIP1/RAD51C/RAD51D were retrieved in 64 patients (1.2%) and alterations in mismatch-repair genes MSH6/MLH1/PMS2/MSH2 were detected in 0.3% of patients. These patients where dominantly referred for familial history of ovarian cancer. Furthermore, germline alterations in PTEN, CDH1 and BLM were observed in 3, 2 and 1 cases respectively. Double heterozygosity for ATM+CHEK2 and for ATM+BRCA2 were both observed in 3 cases. In the patient with triple heterozygosity, co-occurrence of pathogenic variants in BRCA2, ATM and CHEK2 was detected. Conclusions: In a large Belgian cohort of 5422 individuals at risk for HBOC who underwent multi-gene panel testing, a pathogenic or likely pathogenic germline variant was detected in 11,7% of patients, and in 0,46% of patients double or triple heterozygosity for HBOC-variants was observed. Almost 40% of detected variants were alterations beyond BRCA correlated with hereditary breast cancer (CHEK2, ATM, PALB2 and TP53). Given the time-lag to predictive testing in families, a significant rise in healthy carriers with these non-BRCA alterations is expected in the upcoming years. Citation Format: Kevin Punie, Griet Hoste, Griet Van Buggenhout, Ellen Denayer, Hilde Brems, Hilde Peeters, Ann Smeets, Ines Nevelsteen, Patrick Neven, Jan Ardui, Renate Prevos, Machteld Keupers, Chantal Van Ongeval, Giuseppe Floris, Christine Desmedt, Hans Wildiers, Geneviève Michils, Hilde Van Esch, Eric Legius. Germline mutational landscape in 5422 individuals at risk for hereditary breast and ovarian cancer who underwent multi-gene panel testing [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-03.

Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike

There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 A of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.Funding: This work was supported by a grant from the CAMS-Oxford Institute to D.I.S. E.E.F and J.Ren are supported by the Wellcome Trust (101122/Z/13/Z), Y.Z. by Cancer Research UK (C375/A17721) and D.I.S. and E.E.F. by the UK Medical Research Council (MR/N00065X/1). J.H. is supported by a grant from the EPA Cephalosporin Fund. PPUK is funded by the Rosalind Franklin Institute EPSRC Grant no. EP/S025243/1. The National Institute for Health Research Biomedical Research Centre Funding Scheme supports G.R.S. together with the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant number: 2018-I2M-2-002), which also supports D.I.S. G.R.S. is also supported as a Wellcome Trust Senior Investigator (grant 095541/A/11/Z). T.M. is supported by Cancer Research UK grants C20724/A14414 and C20724/A26752 to Christian Siebold. This is a contribution from the UK Instruct-ERIC Centre. The Wellcome Centre for Human Genetics is supported by the Wellcome Trust (grant 090532/Z/09/Z). Virus used for the neutralisation assays was a gift from Julian Druce, Doherty Centre, Melbourne, Australia. Conflict of Interest: The authors declare no competing interests.

&lt;i&gt;Mycobacterium abscessus&lt;/i&gt; Genomic Clusters Span Geography and Patient Groups

Background: Mycobacterium abscessus has emerged as a significant clinical concern following reports that it is readily transmissible in healthcare settings between patients with Cystic Fibrosis (CF). Methods: We analysed consecutive M. abscessus whole-genome sequencing data from England (February-2015 to November-2019) to identify genomically similar isolates. Linkage to a national healthcare usage database was used to investigate possible contacts between patients. Multivariable regression analysis was performed to investigate factors associated with acquisition of a genomically clustered strain (within ≤25 SNPs). Findings: 2297 isolates from 906 patients underwent whole-genome sequencing as part of the routine Public Health England diagnostic service. Of 14 genomic clusters containing ≥10 patients, all but one contained patients with CF and patients without CF. Patients with CF were equally likely to have clustered isolates as those without CF. High-density phylogenetic clusters were randomly distributed over a wide geographical area. The majority of isolates with a closest genetic neighbour consistent with potential transmission had no identifiable relevant epidemiological contacts. Having a clustered isolate was independently associated with increasing age but not time spent as an hospital inpatient/outpatient. We identified two CF sibling pairs with genetically highly divergent isolates and 1 pair with closely related isolates, as well as 25 uninfected presumed household contacts with CF. Interpretation: Previously identified widely disseminated dominant clones of M. abscessus are not restricted to CF patients and occur in other chronic respiratory diseases. Our analysis does not support person-to-person transmission as an important mechanism in M. abscessus dissemination at a national level in England. Funding Statement: The research was supported by the National Institute for Health Research (NIHR) Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance (HPRU 2012-10041) at the University of Oxford in partnership with Public Health England (PHE) and by Oxford NIHR Biomedical Research Centre. T Peto, AS Walker and DW Crook are NIHR Senior Investigators. Computation used the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute supported by Health Data Research UK and the NIHR Oxford Biomedical Research Centre. The report presents independent research funded by NIHR. Declaration of Interests: SL is supported by a Medical Research Council Clinical Research Training Fellowship. DWE declares lecture fees from Gilead, outside the submitted work. TMW is a Wellcome Trust Clinical Career Development Fellow (214560/Z/18/Z). The remaining authors declare no conflicts of interest. Ethics Approval Statement: This work was carried out as a public health investigation with internal approval from Public Health England and therefore ethics committee approval was not required.

Profile of Aravinda Chakravarti

Many of the genetic variations uncovered by genome-wide dragnets are bycatch, not primary disease culprits. Separating causality from coincidence in this sea of data is challenging, especially when many of the candidates do not reside in the coding portion of the genome. Aravinda Chakravarti, director of New York University’s Center for Human Genetics and Genomics, has made a career of not just cutting through the noise but anticipating it, thanks to his training in statistics and genetics. In his Inaugural Article, Chakravarti, who was elected to the National Academy of Sciences in 2015, outlines a systematic process for determining causality in genome-wide association studies. He examines variants of regulatory elements that control gene expression by focusing on variation in cis -regulatory elements that target the SCN5A gene, which is implicated in sudden cardiac death (1). Aravinda Chakravarti. Image courtesy of Sumantra Chatterjee (New York University School of Medicine, New York). Born in Calcutta, India in 1954, just seven years after the country gained independence, Chakravarti remembers a mood of optimism and a sense of possibility. “I grew up in India at a very magical time.” Only in hindsight would Chakravarti call his family’s financial circumstances poor. “I had every kind of material to read, from national newspapers to glossy magazines to books,” he says. “I grew up in a house where reading was very valued, and curiosity was valued.” Chakravarti attended a local high school run by Methodist missionaries that offered advanced science courses. After high school, Chakravarti applied to a plethora of colleges. “I don’t know that I had a particular sense of direction,” he says. That changed as soon as Chakravarti saw the first page of the Indian Statistical Institute’s entrance examination, which contained two 10-digit numbers. The question asked which of five possible answers resulted from …

Estimating the Hidden Burden of Iron Deficiency Among African Children

Background: Iron deficiency (ID) is a major public health burden in African children and accurate prevalence estimates are important for effective nutritional interventions. However, ID may be commonly underestimated in Africa because most measures of iron status are altered by inflammation and infections such as malaria. Methods: We assayed iron and inflammatory biomarkers in 4,853 children aged 0-8 years from Kenya, Uganda, Burkina Faso, South Africa, and The Gambia. We described iron status and its relationship with age, sex, malaria parasitaemia, inflammation, stunting, underweight, and wasting. We defined ID using the WHO ferritin-based definition (ferritin 5mg/L)) and then used regression modelling, correcting for inflammation and malaria, to estimate 'true' prevalence of ID. We further investigated the utility of other iron biomarkers in predicting ID. Findings: The prevalence of ID was highest at one year of age and in male children. Stunting and underweight were associated with anaemia and iron deficient erythropoiesis. Inflammation and malaria parasitaemia significantly influenced all iron biomarkers, although transferrin saturation was least affected. The WHO definition of ID did not identify 27·0% of Burkinabe, 21·4% of Kenyan, 20·0% of Ugandan, 16·8% of Gambian, and 8·5% of South African children with 'true' iron deficiency. This 'hidden burden' increased with age and with increasing prevalence of inflammation and malaria. We observed similar prevalences of ID using regression modelling and transferrin saturation <11%. Interpretation: The prevalence of ID may be underestimated in African children using the WHO definition because inflammation and malaria obscure the 'true' burden. Transferrin saturation is least affected by inflammation and malaria and may more accurately determine the burden of ID. Funding Statement: This work was funded by Wellcome (Grant numbers [110255/Z/15/Z to SHA], [202800/Z/16/Z to TNW; 106289/Z/14/Z to AJM; and 064693, 079110, 095778 to AME] and with core awards to the KEMRI-Wellcome Trust Research Programme (203077/Z/16/Z), The Wellcome Centre for Human Genetics (090532/Z/09/Z, 203141) and the Wellcome Sanger Institute (098051, 206194). AJM was also supported by an Oxford University Clinical Academic School Transitional Fellowship and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. JMM was funded through the DELTAS Africa Initiative (DEL-15-003). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)'s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa's Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust [107769/Z/10/Z] and the UK government. The Gambian studies were supported by National Institute of Child Health and Development, Bill and Melinda Gates Foundation, core funding MC-A760-5QX00 to the MRC Unit The Gambia/MRC International Nutrition Group by the UK MRC and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement. This project also received funding from the European Research Council (ERC) under the European Union’s (Seventh Framework Programme [FP7/2007-2013]) (Grant agreement No 294557 to AVSH). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Ethical approvals were obtained for Kilifi, Kenya (by the Scientific Ethics Review Unit of the Kenya Medical Research Institute (KEMRI/SERU/CGMR-C/046/3257/2983)); Entebbe, Uganda (locally by the Uganda Virus Research Institute (GC/127/12/07/32) and Uganda National Council for Science and Technology (MV625), and in the UK by the London School of Hygiene and Tropical Medicine (A340) and Oxford Tropical Research (OTR) (39-12, 42-14 and 37-15) Ethics Committees); Banfora, Burkina Faso (by Ministere de la Recherche Scientifique et de l’Innovation in Burkina Faso (2014-12-151) and the OTR Ethics Committees (41-12)); Soweto, South Africa (by the University of Witwatersrand Human Research (M130714) and the OTR Ethics Committees (1042-13 and 42-14)); and West Kiang, The Gambia (by the Gambian Government/Medical Research Council Ethics Committee (874/830)).

Abstract LB-239: Studying effects of disease associated polymorphism on a transcriptional pathway: A case study in renal cell cancer

Abstract Genome-wide association studies (GWAS) are an important way of identifying germline polymorphisms that are associated with a specific trait or disease. However, characterizing the functional effects of disease-associated variants is challenging since many are located in intergenic regions of the genome. We have established a methodological framework to study the functional effects of disease-associated polymorphisms by examining their effect on disease- specific transcriptional pathways. Based on the most recent meta-analysis of GWAS in renal cell cancer (RCC), we show that RCC- associated polymorphisms are statistically enriched for binding sites of the transcriptional enhancer, hypoxia-inducible factor (HIF), as identified by ChIP-seq analysis in kidney cancer cells. HIF orchestrates a major transcriptional pathway and is constitutively activated in kidney cancer through loss of the von Hippel-Lindau (VHL) tumor suppressor. At these loci, RCC-associated polymorphisms quantitatively affect HIF binding in allele-specific assays as well as expression of the HIF target genes in eQTL analysis. Furthermore, the association between HIF-binding sites and GWAS signal extends to loci below conventional levels of genome-wide significance thereby implicating these sub-threshold loci in the pathogenesis of RCC. In addition we also observe enrichment of RCC-associated polymorphisms close to, but not directly overlapping HIF-binding sites suggesting that they may alter HIF-target gene expression by mechanisms other than directly affecting HIF-binding. As well as emphasizing the importance of the HIF transcriptional pathway in kidney cancer, this analysis helps distinguish HIF key targets that help drive the pathogenesis of RCC from those that are simply co-activated as a consequence of pathway activation. Although focused on the HIF pathway in kidney cancer, this work also forms a paradigm for studying the effect of non-coding variants linked to other cancers originating from other tissue types. Financial support: The Ludwig Institute for Cancer Research, Cancer Research UK (A16016), National Institute for Health Research (NIHR-RP-2016-06-004), the Wellcome Trust (088182/Z/09/Z, 078333/Z/05/Z, WT091857MA), the Deanship of Scientific Research (DSR), Department of Nephrology and Hypertension, Universitätsklinikum Erlangen und Friedrich-Alexander-University Erlangen- Nürnberg, Ulmenweg 18, 91054 Erlangen, Germany. King Abdulaziz University, Ministry of High Education for Saudi Arabia. High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics is funded by the Wellcome Trust (090532/Z/09/Z). Citation Format: Virginia Schmid, David Mole. Studying effects of disease associated polymorphism on a transcriptional pathway: A case study in renal cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-239.

Stellate Transcriptomics Reveal Consistent Patterns of Gene Expression Across Species

IntroductionSympathetic hyperactivity is a key feature of human hypertension as well as animal models of hypertension and cardiovascular disease. Sympathetic hyperactivity can result from cell‐autonomous changes in neurons that innervate cardiovascular target tissues. Stellate ganglia are of particular interest because the majority of sympathetic neurons innervating the heart reside there. Transcriptomics provides a powerful tool for identifying genomic changes that contribute to sympathetic dysfunction in disease.AimTo compare the transcriptomes of healthy stellate ganglia from adult rat and mouse.MethodRNA‐sequencing was carried out on individual stellate ganglia from 16‐week male Wistar rats or 18‐week male C57Bl6J mice. Mouse samples were depleted of ribosomal RNA prior to library preparation, while the rat library was prepared used OligoDT to enrich for mRNAs. Sequencing was carried out in an Illumina HiSeq 4000 at the High‐Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (Oxford) or the Massively Parallel Sequencing Shared Resource (OHSU). The rat transcriptome was aligned to the reference genome and transcripts were quantified using the Salmon‐DESeq2 method, which relies on exon matching. The mouse transcriptome was aligned to the reference genome by the OHSU Bioinformatics &amp; Computational Biology Core using STAR, which includes non‐coding RNAs.ResultsA comparison of these independent analyses of gene expression reveals a significant degree of consistency regarding the most highly expressed genes, despite differences in the data processing pipeline. Genes closely associated with sympathetic neuron function were among the most highly expressed genes in both rat and mouse. We identified high levels of genes encoding proteins required for norepinephrine synthesis tyrosine hydroxylase (Th), L‐DOPA decarboxylase (Ddc), and dopamine beta hydroxylase (dbh), as well as genes encoding transporters for vesicular packaging (slc18a2, vesicular monoamine transporter 2) and removal of extracellular catecholamines (slc6a2, norepinephrine transporter, NET). High levels of mRNA encoding the sympathetic co‐transmitter Neuropeptide Y (Npy) were also observed.Genes encoding the Nerve Growth Factor receptors Ngfr (p75 neurotrophin receptor) and Trk1 (TrkA tyrosine kinase receptor) were abundantly expressed in rat and mouse stellate ganglia. Genes encoding structural proteins such as actins and tubulins were abundant in both species, as were genes encoding proteins involved in synaptic transmission including Snap25 (synaptosomal‐associated protein 25), Syt1 (synaptotagmin 1), and several nicotinic acetylcholine receptor subunits. The details of transcript counts differed between the two methods but the overall pattern of gene expression between species was quite consistent.ConclusionThese data will provide a normal baseline for identification of changes that occur in diseases associated with sympathetic over‐activity such as hypertension and cardiovascular disease.Support or Funding InformationNIH OT2TR001984, OHSU School of Medicine Dean's Fund, The Wellcome TrustThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.