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1.
JAMA Cardiol ; 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33326012

RESUMO

Importance: Sequencing studies have identified causal genetic variants for distinct subtypes of heart failure (HF) such as hypertrophic or dilated cardiomyopathy. However, the role of rare, high-impact variants in HF, for which ischemic heart disease is the leading cause, has not been systematically investigated. Objective: To assess the contribution of rare variants to all-cause HF with and without reduced left ventricular ejection fraction. Design, Setting, and Participants: This was a retrospective analysis of clinical trials and a prospective epidemiological resource (UK Biobank). Whole-exome sequencing of patients with HF was conducted from the Candesartan in Heart Failure-Assessment of Reduction in Mortality and Morbidity (CHARM) and Controlled Rosuvastatin Multinational Trial in Heart Failure (CORONA) clinical trials. Data were collected from March 1999 to May 2003 for the CHARM studies and September 2003 to July 2007 for the CORONA study. Using a gene-based collapsing approach, the proportion of patients with HF and controls carrying rare and presumed deleterious variants was compared. The burden of pathogenic variants in known cardiomyopathy genes was also investigated to assess the diagnostic yield. Exome sequencing data were generated between January 2018 and October 2018, and analysis began October 2018 and ended April 2020. Main Outcomes and Measures: Odds ratios and P values for genes enriched for rare and presumed deleterious variants in either patients with HF or controls and diagnostic yield of pathogenic variants in known cardiomyopathy genes. Results: This study included 5942 patients with HF and 13 156 controls. The mean (SD) age was 68.9 (9.9) years and 4213 (70.9%) were male. A significant enrichment of protein-truncating variants in the TTN gene (P = 3.35 × 10-13; odds ratio, 2.54; 95% CI, 1.96-3.31) that was further increased after restriction to variants in exons constitutively expressed in the heart (odds ratio, 4.52; 95% CI, 3.10-6.68). Validation using UK Biobank data showed a similar enrichment (odds ratio, 4.97; 95% CI, 3.94-6.19 after restriction). In the clinical trials, 201 of 5916 patients with HF (3.4%) had a pathogenic or likely pathogenic cardiomyopathy variant implicating 21 different genes. Notably, 121 of 201 individuals (60.2%) had ischemic heart disease as the clinically identified etiology for the HF. Individuals with HF and preserved ejection fraction had only a slightly lower yield than individuals with midrange or reduced ejection fraction (20 of 767 [2.6%] vs 15 of 392 [3.8%] vs 166 of 4757 [3.5%]). Conclusions and Relevance: An increased burden of diagnostic mendelian cardiomyopathy variants in a broad group of patients with HF of mostly ischemic etiology compared with controls was observed. This work provides further evidence that mendelian genetic conditions may represent an important subset of complex late-onset diseases such as HF, irrespective of the clinical presentation.

2.
Artigo em Inglês | MEDLINE | ID: mdl-33125268

RESUMO

Background - Spontaneous coronary artery dissection (SCAD) occurs when an epicardial coronary artery is narrowed or occluded by an intramural hematoma. SCAD mainly affects women and is associated with pregnancy and systemic arteriopathies, particularly fibromuscular dysplasia. Variants in several genes, such as those causing connective tissue disorders, have been implicated; however, the genetic architecture is poorly understood. Here, we aim to better understand the diagnostic yield of rare variant genetic testing among a cohort of SCAD survivors and to identify genes or gene-sets that have a significant enrichment of rare variants. Methods - We sequenced a cohort of 384 SCAD survivors from the UK, alongside 13,722 UK Biobank controls and a validation cohort of 92 SCAD survivors. We performed a research diagnostic screen for pathogenic variants, and exome-wide and gene-set rare variant collapsing analyses. Results - The majority of patients within both cohorts are female, 29% of the study cohort and 14% validation cohort have a remote arteriopathy. Four cases across the two cohorts had a diagnosed connective tissue disorder. We identified pathogenic or likely pathogenic variants in seven genes (PKD1, COL3A1, SMAD3, TGFB2, LOX, MYLK, and YY1AP1) in 14/384 cases in the study cohort and in 1/92 cases in the validation cohort. In our rare variant collapsing analysis, PKD1 was the highest ranked gene and several functionally plausible genes were enriched for rare variants, although no gene achieved study-wide statistical significance. Gene-set enrichment analysis suggested a role for additional genes involved in renal function. Conclusions - By studying the largest sequenced cohort of SCAD survivors we demonstrate that, based on current knowledge, only a small proportion have a pathogenic variant that could explain their disease. Our findings strengthen the overlap between SCAD and renal and connective tissue disorders and we highlight several new genes for future validation.

3.
Blood ; 136(17): 1956-1967, 2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-32693407

RESUMO

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.

5.
Nature ; 583(7814): 90-95, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499645

RESUMO

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 malignancies1-3. 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 (BeviMed4) 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.


Assuntos
Doenças da Imunodeficiência Primária/genética , Sequenciamento Completo do Genoma , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Teorema de Bayes , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Doenças da Imunodeficiência Primária/diagnóstico , Doenças da Imunodeficiência Primária/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Fatores de Transcrição/genética
6.
Nature ; 583(7814): 96-102, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581362

RESUMO

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 discovered1. 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 participants2, 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.


Assuntos
Internacionalidade , Programas Nacionais de Saúde , Doenças Raras/diagnóstico , Doenças Raras/genética , Sequenciamento Completo do Genoma , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Bases de Dados Factuais , Eritrócitos/metabolismo , Fator de Transcrição GATA1/genética , Humanos , Fenótipo , Locos de Características Quantitativas , Receptores de Trombopoetina/genética , Medicina Estatal , Reino Unido
7.
Clin Immunol ; 215: 108443, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32353633

RESUMO

A 29-year old male with recurrent respiratory and skin infections, anaemia and neutropaenia during childhood required immunoglobulin replacement for antibody deficiency from age 16. He remained relatively well until age 28 when he presented with a two-week history of fatigue, sore throat, fever and productive cough. He was found to have EBV viraemia and splenomegaly and a diagnosis of EBV-driven lymphoproliferative disease was made following bone marrow trephine. Family history was notable with three siblings: a healthy sister and two brothers with anaemia and neutropaenia; one who succumbed to septicaemia secondary to neutropaenic enterocolitis age 5 and another who developed intestinal vasculitis and antibody deficiency and had a successful haemopoetic stem cell transplant. The proband's DNA underwent targeted sequencing of 279 genes associated with immunodeficiency (GRID panel). The best candidates were two ADA2 variants, p.Arg169Gln (R169Q) and p.Asn370Lys (N370K). Sanger sequencing and co-segregation of variants in the parents, unaffected sister and all three affected brothers was fully consistent with compound heterozygous inheritance. Subsequent whole genome sequencing of the proband identified no other potential causal variants. ADA2 activity was consistent with a diagnosis of ADA2 deficiency in affected family members. This is the first description of EBV-driven lymphoproliferative disease in ADA2 deficiency. ADA2 deficiency may cause susceptibility to severe EBV-induced disease and we would recommend that EBV status and viral load is monitored in patients with this diagnosis and allogeneic SCT is considered at an early stage for patients whose ADA2 deficiency is associated with significant complications.

8.
Hum Mutat ; 41(1): 277-290, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31562665

RESUMO

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.

9.
Neurology ; 93(22): e2007-e2020, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31719132

RESUMO

OBJECTIVES: To determine the frequency of rare and pertinent disease-causing variants in small vessel disease (SVD)-associated genes (such as NOTCH3, HTRA1, COL4A1, COL4A2, FOXC1, TREX1, and GLA) 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 NOTCH3 variants in 11 patients, 2 HTRA1 variants in 2 patients, and 1 missense COL4A1 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 HTRA1. A high-throughput sequencing technology platform is an efficient, reliable method to screen for such mutations.


Assuntos
Doenças de Pequenos Vasos Cerebrais/genética , Acidente Vascular Cerebral Lacunar/genética , Idade de Início , Idoso , CADASIL/genética , Angiopatia Amiloide Cerebral Familiar/genética , Doenças do Tecido Conjuntivo/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Enxaqueca com Aura/genética , Mutação
10.
Blood ; 134(23): 2082-2091, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31064749

RESUMO

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.


Assuntos
Transtornos Plaquetários , Hemorragia , Sequenciamento de Nucleotídeos em Larga Escala , Trombose , Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/genética , Feminino , Dosagem de Genes , Hemorragia/diagnóstico , Hemorragia/genética , Hemostasia/genética , Humanos , Masculino , Trombose/diagnóstico , Trombose/genética
11.
Genet Med ; 21(7): 1576-1584, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30531895

RESUMO

PURPOSE: Increasing numbers of genes are being implicated in Mendelian disorders and incorporated into clinical test panels. However, lack of evidence supporting the gene-disease relationship can hinder interpretation. We explored the utility of testing 51 additional genes for hypertrophic cardiomyopathy (HCM), one of the most commonly tested Mendelian disorders. METHODS: Using genome sequencing data from 240 sarcomere gene negative HCM cases and 6229 controls, we undertook case-control and individual variant analyses to assess 51 genes that have been proposed for HCM testing. RESULTS: We found no evidence to suggest that rare variants in these genes are prevalent causes of HCM. One variant, in a single case, was categorized as likely to be pathogenic. Over 99% of variants were classified as a variant of uncertain significance (VUS) and 54% of cases had one or more VUS. CONCLUSION: For almost all genes, the gene-disease relationship could not be validated and lack of evidence precluded variant interpretation. Thus, the incremental diagnostic yield of extending testing was negligible, and would, we propose, be outweighed by problems that arise with a high rate of uninterpretable findings. These findings highlight the need for rigorous, evidence-based selection of genes for clinical test panels.


Assuntos
Cardiomiopatia Hipertrófica/genética , Sarcômeros , Adolescente , Adulto , Idoso , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/patologia , Estudos de Casos e Controles , Feminino , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Sequenciamento Completo do Genoma , Adulto Jovem
14.
Blood ; 127(23): 2791-803, 2016 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-27084890

RESUMO

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.


Assuntos
Transtornos Plaquetários/genética , Predisposição Genética para Doença , Hemorragia/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Trombose/genética , Estudos de Casos e Controles , Variações do Número de Cópias de DNA , Feminino , Estudos de Associação Genética/métodos , Humanos , Masculino , Mutação , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos
15.
Sci Transl Med ; 8(328): 328ra30, 2016 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-26936507

RESUMO

The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.


Assuntos
Osso e Ossos/patologia , Hemorragia/genética , Mutação/genética , Mielofibrose Primária/genética , Trombocitopenia/genética , Quinases da Família src/genética , Animais , Plaquetas/patologia , Células COS , Chlorocebus aethiops , Feminino , Hematopoese , Hemorragia/complicações , Humanos , Masculino , Linhagem , Fenótipo , Mielofibrose Primária/complicações , Trombocitopenia/complicações , Transfecção , Peixe-Zebra
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