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3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Manuka (Leptospermum scoparium) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Manuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 µg/mL LepteridineTM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, LepteridineTM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that LepteridineTM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by LepteridineTM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural LepteridineTM in Manuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate LepteridineTM bioactivity in Manuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.
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Despite significant progress in unraveling the genetic causes of neurodevelopmental disorders (NDDs), a substantial proportion of individuals with NDDs remain without a genetic diagnosis after microarray and/or exome sequencing. Here, we aimed to assess the power of short-read genome sequencing (GS), complemented with long-read GS, to identify causal variants in participants with NDD from the National Institute for Health and Care Research (NIHR) BioResource project. Short-read GS was conducted on 692 individuals (489 affected and 203 unaffected relatives) from 465 families. Additionally, long-read GS was performed on five affected individuals who had structural variants (SVs) in technically challenging regions, had complex SVs, or required distal variant phasing. Causal variants were identified in 36% of affected individuals (177/489), and a further 23% (112/489) had a variant of uncertain significance after multiple rounds of re-analysis. Among all reported variants, 88% (333/380) were coding nuclear SNVs or insertions and deletions (indels), and the remainder were SVs, non-coding variants, and mitochondrial variants. Furthermore, long-read GS facilitated the resolution of challenging SVs and invalidated variants of difficult interpretation from short-read GS. This study demonstrates the value of short-read GS, complemented with long-read GS, in investigating the genetic causes of NDDs. GS provides a comprehensive and unbiased method of identifying all types of variants throughout the nuclear and mitochondrial genomes in individuals with NDD.
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Genoma Humano , Transtornos do Neurodesenvolvimento , Humanos , Genoma Humano/genética , Mapeamento Cromossômico , Sequência de Bases , Mutação INDEL , Transtornos do Neurodesenvolvimento/genéticaRESUMO
New Zealand manuka (Leptospermum scoparium) honey is a premium food product. Unfortunately, its high demand has led to "not true to label" marketed manuka honey. Robust methods are therefore required to determine authenticity. We previously identified three unique nectar-derived proteins in manuka honey, detected as twelve tryptic peptide markers, and hypothesized these could be used to determine authenticity. We invoked a targeted proteomic approach based on parallel reaction-monitoring (PRM) to selectively monitor relative abundance of these peptides in sixteen manuka and twenty six non-manuka honey samples of various floral origin. We included six tryptic peptide markers derived from three bee-derived major royal jelly proteins as potential internal standards. The twelve manuka-specific tryptic peptide markers were present in all manuka honeys with minor regional variation. By comparison, they had negligible presence in non-manuka honeys. Bee-derived peptides were detected in all honeys with similar relative abundance but with sufficient variation precluding their utility as internal standards. Manuka honeys displayed an inverse relationship between total protein content and the ratio between nectar- to bee-derived peptide abundance. This trend reveals an association between protein content on possible nectar processing time by bees. Overall, these findings demonstrate the first successful application of peptide profiling as an alternative and potentially more robust approach for manuka honey authentication.
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Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a central regulator of immunity. TRAF3 is often somatically mutated in B cell malignancies, but its role in human immunity is not defined. Here, in five unrelated families, we describe an immune dysregulation syndrome of recurrent bacterial infections, autoimmunity, systemic inflammation, B cell lymphoproliferation, and hypergammaglobulinemia. Affected individuals each had monoallelic mutations in TRAF3 that reduced TRAF3 expression. Immunophenotyping showed that patients' B cells were dysregulated, exhibiting increased nuclear factor-κB 2 activation, elevated mitochondrial respiration, and heightened inflammatory responses. Patients had mild CD4+ T cell lymphopenia, with a reduced proportion of naïve T cells but increased regulatory T cells and circulating T follicular helper cells. Guided by this clinical phenotype, targeted analyses demonstrated that common genetic variants, which also reduce TRAF3 expression, are associated with an increased risk of B cell malignancies, systemic lupus erythematosus, higher immunoglobulin levels, and bacterial infections in the wider population. Reduced TRAF3 conveys disease risks by driving B cell hyperactivity via intrinsic activation of multiple intracellular proinflammatory pathways and increased mitochondrial respiration, with a likely contribution from dysregulated T cell help. Thus, we define monogenic TRAF3 haploinsufficiency syndrome and demonstrate how common TRAF3 variants affect a range of human diseases.
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Neoplasias , Fator 3 Associado a Receptor de TNF , Autoimunidade/genética , Linfócitos B , Humanos , Mutação , Neoplasias/patologia , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismoRESUMO
The identification of inherited antithrombin deficiency (ATD) is critical to prevent potentially life-threatening thrombotic events. Causal variants in SERPINC1 are identified for up to 70% of cases, the majority being single-nucleotide variants and indels. The detection and characterization of structural variants (SVs) in ATD remain challenging due to the high number of repetitive elements in SERPINC1. Here, we performed long-read whole-genome sequencing on 10 familial and 9 singleton cases with type I ATD proven by functional and antigen assays, who were selected from a cohort of 340 patients with this rare disorder because genetic analyses were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs. This approach resolved, independently of its size or type, all eight SVs detected by multiple ligation-dependent probe amplification, and identified for the first time a complex rearrangement previously misclassified as a deletion. Remarkably, we identified the mechanism explaining ATD in 2 out of 11 cases with previous unknown defect: the insertion of a novel 2.4 kb SINE-VNTR-Alu retroelement, which was characterized by de novo assembly and verified by specific polymerase chain reaction amplification and sequencing in the probands and affected relatives. The nucleotide-level resolution achieved for all SVs allowed breakpoint analysis, which revealed repetitive elements and microhomologies supporting a common replication-based mechanism for all the SVs. Our study underscores the utility of long-read sequencing technology as a complementary method to identify, characterize, and unveil the molecular mechanism of disease-causing SVs involved in ATD, and enlarges the catalogue of genetic disorders caused by retrotransposon insertions.
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Deficiência de Antitrombina III , Retroelementos , Deficiência de Antitrombina III/diagnóstico , Deficiência de Antitrombina III/genética , Antitrombinas , Humanos , Nucleotídeos , Retroelementos/genéticaRESUMO
Proteomics is an emerging tool in food authentication that has not been optimised for honey analysis. In this study, we present a qualitative proteomic analysis of New Zealand manuka (Leptospermum scoparium) honey. A total of fifty bee-derived proteins were identified in the honey, the most predominant being major royal jelly proteins (MRJPs). We also demonstrate for the first time the presence of unique nectar-derived proteins in manuka honey. A total of 17 manuka plant proteins were identified, a-third of which were putative pathogenesis-related proteins. Two proteins involved in drought tolerance were also identified. Twelve candidate peptides were selected as potential authentication markers based on their uniqueness to manuka honey. Nectar analyses confirmed the origin and specificity of these peptides to L. scoparium nectar, thus presenting peptide profiling as a viable and novel approach for manuka honey authentication. Raw data are available via ProteomeXchange with identifier PXD021730.
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Biomarcadores/análise , Leptospermum/química , Peptídeos/análise , Proteômica/métodos , Néctar de Plantas/químicaRESUMO
We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.
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Aspirina , Testes de Função Plaquetária , Plaquetas , Ciclo-Oxigenase 1/genética , Feminino , Humanos , Agregação Plaquetária/genética , Tromboxano A2RESUMO
Background - 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. Methods - 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. Results - Heterozygous, high impact, likely loss-of-function variants in the Kinase Insert Domain Receptor (KDR) 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 KDR 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 KDR. Four additional PAH cases with rare likely loss-of-function variants in KDR were independently identified in the US PAH Biobank cohort with similar phenotypic characteristics. Conclusions - The Bayesian inference approach allowed us to independently validate KDR, 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 KDR 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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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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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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Grânulos Citoplasmáticos/patologia , Heterogeneidade Genética , Síndrome da Plaqueta Cinza , Sistema Imunitário/patologia , Fenótipo , Biópsia , Proteínas Sanguíneas/genética , Estudos de Casos e Controles , Estudos de Coortes , Grânulos Citoplasmáticos/metabolismo , Diagnóstico Diferencial , Frequência do Gene , Estudos de Associação Genética , Síndrome da Plaqueta Cinza/classificação , Síndrome da Plaqueta Cinza/genética , Síndrome da Plaqueta Cinza/imunologia , Síndrome da Plaqueta Cinza/patologia , Humanos , Sistema Imunitário/fisiologia , Doenças do Sistema Imunitário/sangue , Doenças do Sistema Imunitário/diagnóstico , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/patologia , MutaçãoRESUMO
Disclosing secondary findings (SF) from genome sequencing (GS) can alert carriers to disease risk. However, evidence around variant-disease association and consequences of disclosure for individuals and healthcare services is limited. We report on the feasibility of an approach to identification of SF in inherited cardiac conditions (ICC) genes in participants in a rare disease GS study, followed by targeted clinical evaluation. Qualitative methods were used to explore behavioural and psychosocial consequences of disclosure. ICC genes were analysed in genome sequence data from 7203 research participants; a two-stage approach was used to recruit genotype-blind variant carriers and matched controls. Cardiac-focused medical and family history collection and genetic counselling were followed by standard clinical tests, blinded to genotype. Pathogenic ICC variants were identified in 0.61% of individuals; 20 were eligible for the present study. Four variant carriers and seven non-carrier controls participated. One variant carrier had a family history of ICC and was clinically affected; a second was clinically unaffected and had no relevant family history. One variant, in two unrelated participants, was subsequently reclassified as being of uncertain significance. Analysis of qualitative data highlights participant satisfaction with approach, willingness to follow clinical recommendations, but variable outcomes of relatives' engagement with healthcare services. In conclusion, when offered access to SF, many people choose not to pursue them. For others, disclosure of ICC SF in a specialist setting is valued and of likely clinical utility, and can be expected to identify individuals with, and without a phenotype.
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Aconselhamento Genético/psicologia , Cardiopatias Congênitas/genética , Achados Incidentais , Revelação da Verdade , Adulto , Idoso , Estudos de Viabilidade , Feminino , Aconselhamento Genético/métodos , Testes Genéticos/métodos , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/psicologia , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , FenótipoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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BACKGROUND: Thrombomodulin-associated coagulopathy (TM-AC) is a rare bleeding disorder in which a single reported p.Cys537* variant in the thrombomodulin gene THBD causes high plasma thrombomodulin (TM) levels. High TM levels attenuate thrombin generation and delay fibrinolysis. OBJECTIVES: To report the characteristics of pedigree with a novel THBD variant causing TM-AC, and co-inherited deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI). PATIENTS/METHODS: Identification of pathogenic variants in hemostasis genes by next-generation sequencing and case recall for deep phenotyping. RESULTS: Pedigree members with a previously reported THBD variant predicting p.Pro496Argfs*10 and chain truncation in TM transmembrane domain had abnormal bleeding and greatly increased plasma TM levels. Affected cases had attenuated thrombin generation and delayed fibrinolysis similar to previous reported TM_AC cases with THBD p.Cys537*. Coincidentally, some pedigree members also harbored a stop-gain variant in CPB2 encoding TAFI. This reduced plasma TAFI levels but was asymptomatic. Pedigree members with TM-AC caused by the p.Pro496Argfs*10 THBD variant and also TAFI deficiency had a partially attenuated delay in fibrinolysis, but no change in the defective thrombin generation. CONCLUSIONS: These data extend the reported genetic repertoire of TM-AC and establish a common molecular pathogenesis arising from high plasma levels of TM extra-cellular domain. The data further confirm that the delay in fibrinolysis associated with TM-AC is directly linked to increased TAFI activation. The combination of the rare variants in the pedigree members provides a unique genetic model to develop understanding of the thrombin-TM system and its regulation of TAFI.
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Transtornos da Coagulação Sanguínea , Carboxipeptidase B2 , Carboxipeptidase B2/genética , Fibrinólise/genética , Humanos , Linhagem , Trombina , Trombomodulina/genéticaRESUMO
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.
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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 UnidoRESUMO
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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Estudos de Associação Genética , Mutação , Telangiectasia Hemorrágica Hereditária/genética , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/genética , Estudos de Coortes , Análise Mutacional de DNA/métodos , Endoglina/química , Endoglina/genética , Feminino , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Testes Genéticos/métodos , Genômica/métodos , Fator 2 de Diferenciação de Crescimento/química , Fator 2 de Diferenciação de Crescimento/genética , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Modelos Moleculares , Fenótipo , Estudos Retrospectivos , Análise de Sequência de DNA/métodos , Proteína Smad4/química , Proteína Smad4/genética , Telangiectasia Hemorrágica Hereditária/epidemiologia , Telangiectasia Hemorrágica Hereditária/patologiaRESUMO
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.
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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/genéticaRESUMO
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.
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Adenosina Desaminase/deficiência , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/patogenicidade , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Transtornos Linfoproliferativos/complicações , Transtornos Linfoproliferativos/metabolismo , Adulto , Humanos , MasculinoRESUMO
BACKGROUND: The MNS blood group system is defined by three homologous genes: GYPA, GYPB, and GYPE. GYPB encodes for glycophorin B (GPB) carrying S/s and the "universal" antigen U. RBCs of approximately 1% of individuals of African ancestry are U- due to absence of GPB. The U- phenotype has long been attributed to a deletion encompassing GYPB exons 2 to 5 and GYPE exon 1 (GYPB*01N). STUDY DESIGN AND METHODS: Samples from two U-individuals underwent Illumina short read whole genome sequencing (WGS) and Nanopore long read WGS. In addition, two existing WGS datasets, MedSeq (n = 110) and 1000 Genomes (1000G, n = 2535), were analyzed for GYPB deletions. Deletions were confirmed by Sanger sequencing. Twenty known U- donor samples were tested by a PCR assay to determine the specific deletion alleles present in African Americans. RESULTS: Two large GYPB deletions in U- samples of African ancestry were identified: a 110 kb deletion extending left of GYPB (DEL_B_LEFT) and a 103 kb deletion extending right (DEL_B_RIGHT). DEL_B_LEFT and DEL_B_RIGHT were the most common GYPB deletions in the 1000 Genomes Project 669 African genomes (allele frequencies 0.04 and 0.02). Seven additional deletions involving GYPB were seen in African, Admixed American, and South Asian samples. No samples analyzed had GYPB*01N. CONCLUSIONS: The U- phenotype in those of African ancestry is primarily associated with two different complete deletions of GYPB (with intact GYPE). Seven additional less common GYPB deletion backgrounds were found. GYPB*01N, long assumed to be the allele commonly encoding U- phenotypes, appears to be rare.
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Negro ou Afro-Americano/genética , Éxons , Deleção de Genes , Glicoforinas/genética , Sistema do Grupo Sanguíneo MNSs/genética , HumanosRESUMO
Manuka honey is a premium food product with unique antimicrobial bioactivity. Concerns with mislabeled manuka honey require robust assays to determine authenticity. Lepteridine is a Leptospermum-specific fluorescent molecule with potential as an authenticity marker. We describe a mass spectrometry-based assay to measure lepteridine based on an isotopically labeled lepteridine standard. Using this assay, lepteridine concentrations in manuka honey samples strongly correlated with concentrations quantitated by either high-performance liquid chromatography-ultraviolet (HPLC-UV) or fluorescence. A derived minimum lepteridine threshold concentration was compared with the New Zealand regulatory definition for manuka honey to determine "manuka honey" authenticity on a set of commercial samples. Both methods effectively distinguished manuka honey from non-manuka honeys. The regulatory definition excludes lepteridine but otherwise includes the quantification of multiple floral markers together with pollen analysis. Our findings suggest that the quantification of lepteridine alone or in combination with leptosperin could be implemented as an effective screening method to identify manuka honey, likely to achieve an outcome similar to the regulatory definition.
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Several strands of evidence question the dogma that human mitochondrial DNA (mtDNA) is inherited exclusively down the maternal line, most recently in three families where several individuals harbored a 'heteroplasmic haplotype' consistent with biparental transmission. Here we report a similar genetic signature in 7 of 11,035 trios, with allelic fractions of 5-25%, implying biparental inheritance of mtDNA in 0.06% of offspring. However, analysing the nuclear whole genome sequence, we observe likely large rare or unique nuclear-mitochondrial DNA segments (mega-NUMTs) transmitted from the father in all 7 families. Independently detecting mega-NUMTs in 0.13% of fathers, we see autosomal transmission of the haplotype. Finally, we show the haplotype allele fraction can be explained by complex concatenated mtDNA-derived sequences rearranged within the nuclear genome. We conclude that rare cryptic mega-NUMTs can resemble paternally mtDNA heteroplasmy, but find no evidence of paternal transmission of mtDNA in humans.