Publications by authors named "Karyn Megy"

26 Publications

  • Page 1 of 1

Advances in understanding the pathogenesis of hereditary macrothrombocytopenia.

Br J Haematol 2021 Mar 30. Epub 2021 Mar 30.

Department of Biostatistics and Epidemiology, Faculty of Medicine, Imperial College London, London, UK.

Low platelet count, or thrombocytopenia, is a common haematological abnormality, with a wide differential diagnosis, which may represent a clinically significant underlying pathology. Macrothrombocytopenia, the presence of large platelets in combination with thrombocytopenia, can be acquired or hereditary and indicative of a complex disorder. In this review, we discuss the interpretation of platelet count and volume measured by automated haematology analysers and highlight some important technical considerations relevant to the analysis of blood samples with macrothrombocytopenia. We review how large cohorts, such as the UK Biobank and INTERVAL studies, have enabled an accurate description of the distribution and co-variation of platelet parameters in adult populations. We discuss how genome-wide association studies have identified hundreds of genetic associations with platelet count and mean platelet volume, which in aggregate can explain large fractions of phenotypic variance, consistent with a complex genetic architecture and polygenic inheritance. Finally, we describe the large genetic diagnostic and discovery programmes, which, simultaneously to genome-wide association studies, have expanded the repertoire of genes and variants associated with extreme platelet phenotypes. These have advanced our understanding of the pathogenesis of hereditary macrothrombocytopenia and support a future clinical diagnostic strategy that utilises genotype alongside clinical and laboratory phenotype data.
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http://dx.doi.org/10.1111/bjh.17409DOI Listing
March 2021

Whole Genome Interpretation for a Family of Five.

Front Genet 2021 8;12:535123. Epub 2021 Mar 8.

Cambridge Precision Medicine Limited, ideaSpace, University of Cambridge Biomedical Innovation Hub, Cambridge, United Kingdom.

Although best practices have emerged on how to analyse and interpret personal genomes, the utility of whole genome screening remains underdeveloped. A large amount of information can be gathered from various types of analyses via whole genome sequencing including pathogenicity screening, genetic risk scoring, fitness, nutrition, and pharmacogenomic analysis. We recognize different levels of confidence when assessing the validity of genetic markers and apply rigorous standards for evaluation of phenotype associations. We illustrate the application of this approach on a family of five. By applying analyses of whole genomes from different methodological perspectives, we are able to build a more comprehensive picture to assist decision making in preventative healthcare and well-being management. Our interpretation and reporting outputs provide input for a clinician to develop a healthcare plan for the individual, based on genetic and other healthcare data.
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http://dx.doi.org/10.3389/fgene.2021.535123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982663PMC
March 2021

Bayesian Inference Associates Rare Variants with Specific Phenotypes in Pulmonary Arterial Hypertension.

Circ Genom Precis Med 2020 Dec 15. Epub 2020 Dec 15.

Université Paris-Sud, Faculté de Médecine, Université Paris-Saclay & AP-HP, Service de Pneumologie, Centre de référence de l'hypertension pulmonaire & INSERM UMR_S 999, Hôpital Bicêtre, Le Kremlin-Bicêtre, Paris, France.

- Approximately 25% of patients with pulmonary arterial hypertension (PAH) have been found to harbor rare mutations in disease-causing genes. To identify missing heritability in PAH we integrated deep phenotyping with whole-genome sequencing data using Bayesian statistics. - We analyzed 13,037 participants enrolled in the NIHR BioResource - Rare Diseases (NBR) study, of which 1,148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension (PH), we used the Bayesian rare-variant association method BeviMed. - Heterozygous, high impact, likely loss-of-function variants in the Kinase Insert Domain Receptor () gene were strongly associated with significantly reduced transfer coefficient for carbon monoxide (KCO, posterior probability (PP)=0.989) and older age at diagnosis (PP=0.912). We also provide evidence for familial segregation of a rare nonsense variant with these phenotypes. On computed tomographic imaging of the lungs, a range of parenchymal abnormalities were observed in the five patients harboring these predicted deleterious variants in . Four additional PAH cases with rare likely loss-of-function variants in were independently identified in the US PAH Biobank cohort with similar phenotypic characteristics. - The Bayesian inference approach allowed us to independently validate , which encodes for the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), as a novel PAH candidate gene. Furthermore, this approach specifically associated high impact likely loss-of-function variants in the genetically constrained gene with distinct phenotypes. These findings provide evidence for being a clinically actionable PAH gene and further support the central role of the vascular endothelium in the pathobiology of PAH.
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http://dx.doi.org/10.1161/CIRCGEN.120.003155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892262PMC
December 2020

The Polygenic and Monogenic Basis of Blood Traits and Diseases.

Cell 2020 09;182(5):1214-1231.e11

Laboratory of Epidemiology and Population Science, National Institute on Aging/NIH, Baltimore, MD, 21224, USA.

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation.
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http://dx.doi.org/10.1016/j.cell.2020.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482360PMC
September 2020

Development and validation of a universal blood donor genotyping platform: a multinational prospective study.

Blood Adv 2020 08;4(15):3495-3506

British Heart Foundation Cardiovascular Epidemiology Unit, Department of Public Health and Primary Care, University of Cambridge, Cambridge, United Kingdom.

Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non-self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.
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http://dx.doi.org/10.1182/bloodadvances.2020001894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422129PMC
August 2020

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Blood 2020 10;136(17):1956-1967

Service d'Hématologie Biologique, Hospices Civils de Lyon, Lyon, France.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.
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http://dx.doi.org/10.1182/blood.2019004776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582559PMC
October 2020

Whole-genome sequencing of patients with rare diseases in a national health system.

Nature 2020 07 24;583(7814):96-102. Epub 2020 Jun 24.

Department of Medical Genetics, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK.

Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.
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http://dx.doi.org/10.1038/s41586-020-2434-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610553PMC
July 2020

Mutational and phenotypic characterization of hereditary hemorrhagic telangiectasia.

Blood 2020 10;136(17):1907-1918

Department of Haematology, Cambridge Biomedical Campus, University of Cambridge, Cambridge, United Kingdom.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.
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http://dx.doi.org/10.1182/blood.2019004560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717479PMC
October 2020

Whole-genome sequencing of a sporadic primary immunodeficiency cohort.

Nature 2020 07 6;583(7814):90-95. Epub 2020 May 6.

Institute of Immunity and Transplantation, University College London, London, UK.

Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies. Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed) identified multiple new candidate PID-associated genes, including IVNS1ABP. We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of-and interplay between-novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans.
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http://dx.doi.org/10.1038/s41586-020-2265-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334047PMC
July 2020

Large-Scale Whole-Genome Sequencing Reveals the Genetic Architecture of Primary Membranoproliferative GN and C3 Glomerulopathy.

J Am Soc Nephrol 2020 02 9;31(2):365-373. Epub 2020 Jan 9.

Department of Renal Medicine, University College London, London, United Kingdom;

Background: Primary membranoproliferative GN, including complement 3 (C3) glomerulopathy, is a rare, untreatable kidney disease characterized by glomerular complement deposition. Complement gene mutations can cause familial C3 glomerulopathy, and studies have reported rare variants in complement genes in nonfamilial primary membranoproliferative GN.

Methods: We analyzed whole-genome sequence data from 165 primary membranoproliferative GN cases and 10,250 individuals without the condition (controls) as part of the National Institutes of Health Research BioResource-Rare Diseases Study. We examined copy number, rare, and common variants.

Results: Our analysis included 146 primary membranoproliferative GN cases and 6442 controls who were unrelated and of European ancestry. We observed no significant enrichment of rare variants in candidate genes (genes encoding components of the complement alternative pathway and other genes associated with the related disease atypical hemolytic uremic syndrome; 6.8% in cases versus 5.9% in controls) or exome-wide. However, a significant common variant locus was identified at 6p21.32 (rs35406322) (=3.29×10; odds ratio [OR], 1.93; 95% confidence interval [95% CI], 1.53 to 2.44), overlapping the HLA locus. Imputation of HLA types mapped this signal to a haplotype incorporating DQA1*05:01, DQB1*02:01, and DRB1*03:01 (=1.21×10; OR, 2.19; 95% CI, 1.66 to 2.89). This finding was replicated by analysis of HLA serotypes in 338 individuals with membranoproliferative GN and 15,614 individuals with nonimmune renal failure.

Conclusions: We found that HLA type, but not rare complement gene variation, is associated with primary membranoproliferative GN. These findings challenge the paradigm of complement gene mutations typically causing primary membranoproliferative GN and implicate an underlying autoimmune mechanism in most cases.
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http://dx.doi.org/10.1681/ASN.2019040433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003307PMC
February 2020

How common are single gene mutations as a cause for lacunar stroke? A targeted gene panel study.

Neurology 2019 11 12;93(22):e2007-e2020. Epub 2019 Nov 12.

From the Stroke Research Group, Department of Clinical Neurosciences (R.Y.Y.T., M.T., H.S.M.), Department of Haematology (K.M., D.D., S.V.V.D., O.S., R.P.M., W.H.O., S.G., K.D.), and Division of Respiratory Medicine, Department of Medicine (S.G.), University of Cambridge; and NIHR BioResource: Rare Diseases (K.M., S.V.V.D., O.S., R.P.M., W.H.O., S.G., H.S.M.), Biomedical Campus, Cambridge, UK.

Objectives: To determine the frequency of rare and pertinent disease-causing variants in small vessel disease (SVD)-associated genes (such as , , , , , , and ) in cerebral SVD, we performed targeted gene sequencing in 950 patients with younger-onset apparently sporadic SVD stroke using a targeted sequencing panel.

Methods: We designed a high-throughput sequencing panel to identify variants in 15 genes (7 known SVD genes, 8 SVD-related disorder genes). The panel was used to screen a population of 950 patients with younger-onset (≤70 years) MRI-confirmed SVD stroke, recruited from stroke centers across the United Kingdom. Variants were filtered according to their frequency in control databases, predicted effect, presence in curated variant lists, and combined annotation dependent depletion scores. Whole genome sequencing and genotyping were performed on a subset of patients to provide a direct comparison of techniques. The frequency of known disease-causing and pertinent variants of uncertain significance was calculated.

Results: We identified previously reported variants in 14 patients (8 cysteine-changing variants in 11 patients, 2 variants in 2 patients, and 1 missense variant in 1 patient). In addition, we identified 29 variants of uncertain significance in 32 patients.

Conclusion: Rare monogenic variants account for about 1.5% of younger onset lacunar stroke. Most are cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy variants, but the second most common gene affected is A high-throughput sequencing technology platform is an efficient, reliable method to screen for such mutations.
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http://dx.doi.org/10.1212/WNL.0000000000008544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913325PMC
November 2019

Next-generation sequencing for the diagnosis of MYH9-RD: Predicting pathogenic variants.

Hum Mutat 2020 01 15;41(1):277-290. Epub 2019 Oct 15.

School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK.

The heterogeneous manifestations of MYH9-related disorder (MYH9-RD), characterized by macrothrombocytopenia, Döhle-like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE-BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS). We identified 50 patients with a rare variant in MYH9. All patients had macrothrombocytes and all except two had thrombocytopenia. Some degree of bleeding diathesis was reported in 41 of the 50 patients. Eleven patients presented hearing impairment, three renal failure and two elevated liver enzymes. Among the 28 rare variants identified in MYH9, 12 were novel. HTS was instrumental in diagnosing 23 patients (46%). Our results confirm the clinical heterogeneity of MYH9-RD and show that, in the presence of an unclassified platelet disorder with macrothrombocytes, MYH9-RD should always be considered. A HTS-based strategy is a reliable method to reach a conclusive diagnosis of MYH9-RD in clinical practice.
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http://dx.doi.org/10.1002/humu.23927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972977PMC
January 2020

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

Blood 2019 12;134(23):2070-2081

National Institute for Health Research (NIHR) BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.
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http://dx.doi.org/10.1182/blood.2019000782DOI Listing
December 2019

De novo variant in tyrosine kinase SRC causes thrombocytopenia: case report of a second family.

Platelets 2019 17;30(7):931-934. Epub 2019 Jun 17.

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven , Leuven , Belgium.

A germline heterozygous gain-of-function .E527K variant in tyrosine kinase SRC was previously found to cause thrombocytopenia, myelofibrosis, bleeding, bone pathologies, premature edentulism and mild facial dysmorphia in nine patients of a single pedigree. Because of this variant, SRC loses its self-inhibitory capacity, causing constitutively active SRC expression in platelets. These patients have fewer and heterogeneous-sized platelets that are hyporeactive to collagen. We now report a 5-year-old girl with syndromic thrombocytopenia due to the same SRC-E527K variant that occurs . A bone marrow biopsy, blood smear analysis, platelet aggregations, flow cytometric analysis of -selectin, SRC expression and tyrosine phosphorylation studies were performed to confirm the similarities between the two families. This study strengthens our previous finding that hyperactive SRC kinase results in mild platelet dysfunction and thrombocytopenia with hypogranular platelets and further expands the clinical description of this syndrome to improve early recognition.
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http://dx.doi.org/10.1080/09537104.2019.1628197DOI Listing
January 2020

Diagnostic high-throughput sequencing of 2396 patients with bleeding, thrombotic, and platelet disorders.

Blood 2019 12;134(23):2082-2091

East Midlands and East of England Genomic Laboratory Hub, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, United Kingdom.

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.
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http://dx.doi.org/10.1182/blood.2018891192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993014PMC
December 2019

Whole genome sequencing reveals that genetic conditions are frequent in intensively ill children.

Intensive Care Med 2019 05 7;45(5):627-636. Epub 2019 Mar 7.

School of Clinical Medicine, University of Cambridge, Cambridge Biomedical Campus, Cambridge, CB2 0SP, UK.

Purpose: With growing evidence that rare single gene disorders present in the neonatal period, there is a need for rapid, systematic, and comprehensive genomic diagnoses in ICUs to assist acute and long-term clinical decisions. This study aimed to identify genetic conditions in neonatal (NICU) and paediatric (PICU) intensive care populations.

Methods: We performed trio whole genome sequence (WGS) analysis on a prospective cohort of families recruited in NICU and PICU at a single site in the UK. We developed a research pipeline in collaboration with the National Health Service to deliver validated pertinent pathogenic findings within 2-3 weeks of recruitment.

Results: A total of 195 families had whole genome analysis performed (567 samples) and 21% received a molecular diagnosis for the underlying genetic condition in the child. The phenotypic description of the child was a poor predictor of the gene identified in 90% of cases, arguing for gene agnostic testing in NICU/PICU. The diagnosis affected clinical management in more than 65% of cases (83% in neonates) including modification of treatments and care pathways and/or informing palliative care decisions. A 2-3 week turnaround was sufficient to impact most clinical decision-making.

Conclusions: The use of WGS in intensively ill children is acceptable and trio analysis facilitates diagnoses. A gene agnostic approach was effective in identifying an underlying genetic condition, with phenotypes and symptomatology being primarily used for data interpretation rather than gene selection. WGS analysis has the potential to be a first-line diagnostic tool for a subset of intensively ill children.
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http://dx.doi.org/10.1007/s00134-019-05552-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483967PMC
May 2019

Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing.

Genome Med 2018 12 7;10(1):95. Epub 2018 Dec 7.

Department of Haematology, University of Cambridge, NHS Blood and Transplant Centre, Cambridge, CB2 0PT, UK.

Background: Studies have shown that complex structural variants (cxSVs) contribute to human genomic variation and can cause Mendelian disease. We aimed to identify cxSVs relevant to Mendelian disease using short-read whole-genome sequencing (WGS), resolve the precise variant configuration and investigate possible mechanisms of cxSV formation.

Methods: We performed short-read WGS and analysis of breakpoint junctions to identify cxSVs in a cohort of 1324 undiagnosed rare disease patients. Long-read WGS and gene expression analysis were used to resolve one case.

Results: We identified three pathogenic cxSVs: a de novo duplication-inversion-inversion-deletion affecting ARID1B, a de novo deletion-inversion-duplication affecting HNRNPU and a homozygous deletion-inversion-deletion affecting CEP78. Additionally, a de novo duplication-inversion-duplication overlapping CDKL5 was resolved by long-read WGS demonstrating the presence of both a disrupted and an intact copy of CDKL5 on the same allele, and gene expression analysis showed both parental alleles of CDKL5 were expressed. Breakpoint analysis in all the cxSVs revealed both microhomology and longer repetitive elements.

Conclusions: Our results corroborate that cxSVs cause Mendelian disease, and we recommend their consideration during clinical investigations. We show that resolution of breakpoints can be critical to interpret pathogenicity and present evidence of replication-based mechanisms in cxSV formation.
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http://dx.doi.org/10.1186/s13073-018-0606-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286558PMC
December 2018

Comprehensive Cancer-Predisposition Gene Testing in an Adult Multiple Primary Tumor Series Shows a Broad Range of Deleterious Variants and Atypical Tumor Phenotypes.

Am J Hum Genet 2018 07 14;103(1):3-18. Epub 2018 Jun 14.

North East Thames Regional Genetics Service, Great Ormond Street Hospital for Children, London WC1N 3JH, UK.

Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-affected individuals for variants in cancer-predisposition genes (CPGs) is mostly targeted by tumor type. We ascertained pre-assessed MPT individuals (with at least two primary tumors by age 60 years or at least three by 70 years) from genetics centers and performed whole-genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment and molecular investigations, pathogenic variants in moderate- and high-risk CPGs were detected in 67/440 (15.2%) probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies, including low-frequency structural variants (6/440 [1.4%] probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant), the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice-site CPG variants and at least one discordant tumor type was significantly higher than in a control population (χ = 43.642; p ≤ 0.0001). 2/67 (3%) probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Together with variant detection rates from a previous series of similarly ascertained MPT-affected individuals, the present results suggest that first-line comprehensive CPG analysis in an MPT cohort referred to clinical genetics services would detect a deleterious variant in about a third of individuals.
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http://dx.doi.org/10.1016/j.ajhg.2018.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037202PMC
July 2018

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

Am J Hum Genet 2017 01 29;100(1):75-90. Epub 2016 Dec 29.

NIHR BioResource - Rare Diseases, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge CB2 0QQ, UK; Great Ormond Street Hospital for Children, Great Ormond Street, London WC1N 3JH, UK.

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.
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http://dx.doi.org/10.1016/j.ajhg.2016.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223092PMC
January 2017

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.

Cell 2016 11;167(5):1415-1429.e19

Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK; Department of Statistics, University of Oxford, 1 South Parks Road, Oxford OX1 3TG, UK.

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.
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http://dx.doi.org/10.1016/j.cell.2016.10.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300907PMC
November 2016

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Blood 2016 06 15;127(23):2791-803. Epub 2016 Apr 15.

Department of Haematology, University of Cambridge, National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom;

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.
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http://dx.doi.org/10.1182/blood-2015-12-688267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016734PMC
June 2016

ArrayExpress update--simplifying data submissions.

Nucleic Acids Res 2015 Jan 31;43(Database issue):D1113-6. Epub 2014 Oct 31.

European Molecular Biology Laboratory, European Bioinformatics Institute, EMBL-EBI, Wellcome Trust Genome Campus, Hinxton, CB10 1SD, UK

The ArrayExpress Archive of Functional Genomics Data (http://www.ebi.ac.uk/arrayexpress) is an international functional genomics database at the European Bioinformatics Institute (EMBL-EBI) recommended by most journals as a repository for data supporting peer-reviewed publications. It contains data from over 7000 public sequencing and 42,000 array-based studies comprising over 1.5 million assays in total. The proportion of sequencing-based submissions has grown significantly over the last few years and has doubled in the last 18 months, whilst the rate of microarray submissions is growing slightly. All data in ArrayExpress are available in the MAGE-TAB format, which allows robust linking to data analysis and visualization tools and standardized analysis. The main development over the last two years has been the release of a new data submission tool Annotare, which has reduced the average submission time almost 3-fold. In the near future, Annotare will become the only submission route into ArrayExpress, alongside MAGE-TAB format-based pipelines. ArrayExpress is a stable and highly accessed resource. Our future tasks include automation of data flows and further integration with other EMBL-EBI resources for the representation of multi-omics data.
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http://dx.doi.org/10.1093/nar/gku1057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383899PMC
January 2015

Comparative genomics allows the discovery of cis-regulatory elements in mosquitoes.

Proc Natl Acad Sci U S A 2009 Mar 11;106(9):3053-8. Epub 2009 Feb 11.

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.

The discovery and mapping of cis-regulatory elements is important for understanding regulation of gene transcription in mosquito vectors of human diseases. Genome sequence data are available for 3 species, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus (Diptera: Culicidae), representing 2 subfamilies (Culicinae and Anophelinae) that are estimated to have diverged 145 to 200 million years ago. Comparative genomics tools were used to screen genomic DNA fragments located in the 5'-end flanking regions of orthologous genes. These analyses resulted in the identification of 137 sequences, designated "mosquito motifs," 7 to 9 nucleotides in length, representing 18 families of putative cis-regulatory elements conserved significantly among the 3 species when compared to the fruit fly, Drosophila melanogaster. Forty-one of the motifs were implicated previously in experiments as sites for binding transcription factors or functioning in the regulation of mosquito gene expression. Further analyses revealed associations between specific motifs and expression profiles, particularly in those genes that show increased or decreased mRNA abundance in females following a blood meal, and those accumulating transcription products exclusively or preferentially in the midgut, fat bodies, or ovaries. These results validate the methodology and support a relationship between the discovered motifs and the conservation of hematophagy in mosquitoes.
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http://dx.doi.org/10.1073/pnas.0813264106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2640218PMC
March 2009

Genome sequence of Aedes aegypti, a major arbovirus vector.

Science 2007 Jun 17;316(5832):1718-23. Epub 2007 May 17.

Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA.

We present a draft sequence of the genome of Aedes aegypti, the primary vector for yellow fever and dengue fever, which at approximately 1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae. Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of approximately 4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster. Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of approximately 2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species.
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http://dx.doi.org/10.1126/science.1138878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2868357PMC
June 2007