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1.
Hum Mutat ; 2019 Sep 28.
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.

3.
Blood ; 2019 May 07.
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 multi-disciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2,396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbour variants associated with rare bleeding, thrombotic or platelet disorders (BTPD). 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 and intronic variants, as Pathogenic, Likely Pathogenic or Variants of Uncertain Significance. Half (50.9%) of these variants are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPD. 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.

4.
Clin Immunol ; 203: 23-27, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30953794

RESUMO

Genetic studies are identifying an increasing number of monogenic causes of Common Variable Immunodeficiency (CVID). Pathogenic variants in the C-terminus of NFKB2 have been identified in the subset of CVID patients whose immunodeficiency is associated with ectodermal dysplasia and central adrenal insufficiency. We describe 2 unrelated CVID pedigrees with 4 cases of pathogenic stop gain variants (c.1903C > T) in the ankyrin repeat domain (ARD) of NF-κB2, leading to a premature truncation of the protein at p.Arg635Term (R635X). By immunophenotyping and functional ex vivo B- and T-cell experiments we characterized the variant by reduced class-switched memory B-cell counts and immature plasmablasts, unable to produce IgG and IgA. Features of a poor proliferative T-cell response and reduced expansion of CD4+CXCR5+ T cells was only observed in the two clinically affected index cases without any clear clinical correlate. In conclusion, pathogenic stop variants in the ARD of NFKB2 can cause 'infection-only' CVID with an abnormal B-cell phenotype and a variable clinical penetrance.

5.
J Allergy Clin Immunol ; 142(4): 1285-1296, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29477724

RESUMO

BACKGROUND: The genetic cause of primary immunodeficiency disease (PID) carries prognostic information. OBJECTIVE: We conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource-Rare Diseases cohort. METHODS: In the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense, and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n = 390) in the cohort. Amino acid substitutions predicted to be pathogenic were assessed by means of analysis of structural protein data. Immunophenotyping, immunoblotting, and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype cosegregation analyses. RESULTS: Both sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B-lymphocyte differentiation. Detailed assessment of B-lymphocyte numbers, phenotype, and function identifies the presence of an increased CD21low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases. CONCLUSION: We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.

6.
Orphanet J Rare Dis ; 12(1): 101, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28545593

RESUMO

BACKGROUND: Several genetic defects have been identified in the glycosylphosphatidylinositol (GPI) anchor synthesis, including mutations in PIGO encoding phosphatidylinositol glycan anchor biosynthesis class O protein. These defects constitute a subgroup of the congenital disorders of glycosylation (CDG). Seven patients from five families have been reported carrying variants in PIGO that cause an autosomal recessive syndrome characterised by dysmorphism, psychomotor disability, epilepsy and hyperphosphatasemia. METHODS: Whole exome sequencing was performed in a boy with dysmorphism, psychomotor disability, epilepsy, palmoplantar keratoderma, hyperphosphatasemia and platelet dysfunction without a clinical bleeding phenotype. RESULTS: Two novel variants in PIGO were detected. The missense variant encoding p. His871Pro was inherited from the boy's father while the frameshift variant encoding p. Arg604ProfsTer40 was maternally inherited. CONCLUSION: A boy with two novel PIGO variants is reported. The skin phenotype and platelet dysfunction in this patient have not been described in previously reported patients with PIGO deficiency but it is of course uncertain whether these are caused by this disorder. The literature on PIGO deficiency is reviewed.


Assuntos
Ceratodermia Palmar e Plantar/diagnóstico , Ceratodermia Palmar e Plantar/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Mutação/genética , Adolescente , Glicosilfosfatidilinositóis/deficiência , Glicosilfosfatidilinositóis/genética , Humanos , Ceratodermia Palmar e Plantar/metabolismo , Masculino
7.
Blood ; 129(4): 520-524, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28064200

RESUMO

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibß, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.


Assuntos
Plaquetas/metabolismo , Hemorragia/genética , Mutação , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Trombocitopenia/genética , Alelos , Plaquetas/patologia , Estudos de Casos e Controles , Feminino , Expressão Gênica , Genes Dominantes , Genoma Humano , Hemorragia/diagnóstico , Hemorragia/metabolismo , Hemorragia/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Contagem de Plaquetas , Trombocitopenia/diagnóstico , Trombocitopenia/metabolismo , Trombocitopenia/patologia
8.
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
9.
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 , Cercopithecus aethiops , Feminino , Hematopoese , Hemorragia/complicações , Humanos , Masculino , Linhagem , Fenótipo , Mielofibrose Primária/complicações , Trombocitopenia/complicações , Transfecção , Peixe-Zebra
10.
Blood ; 127(23): 2903-14, 2016 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-26912466

RESUMO

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Perda Auditiva/genética , Mutação , Trombocitopenia/genética , Células A549 , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Células Cultivadas , Criança , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Perda Auditiva/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único , Síndrome , Trombocitopenia/complicações , Adulto Jovem
11.
Genome Med ; 7(1): 36, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25949529

RESUMO

BACKGROUND: Heritable bleeding and platelet disorders (BPD) are heterogeneous and frequently have an unknown genetic basis. The BRIDGE-BPD study aims to discover new causal genes for BPD by high throughput sequencing using cluster analyses based on improved and standardised deep, multi-system phenotyping of cases. METHODS: We report a new approach in which the clinical and laboratory characteristics of BPD cases are annotated with adapted Human Phenotype Ontology (HPO) terms. Cluster analyses are then used to characterise groups of cases with similar HPO terms and variants in the same genes. RESULTS: We show that 60% of index cases with heritable BPD enrolled at 10 European or US centres were annotated with HPO terms indicating abnormalities in organ systems other than blood or blood-forming tissues, particularly the nervous system. Cases within pedigrees clustered closely together on the bases of their HPO-coded phenotypes, as did cases sharing several clinically suspected syndromic disorders. Cases subsequently found to harbour variants in ACTN1 also clustered closely, even though diagnosis of this recently described disorder was not possible using only the clinical and laboratory data available to the enrolling clinician. CONCLUSIONS: These findings validate our novel HPO-based phenotype clustering methodology for known BPD, thus providing a new discovery tool for BPD of unknown genetic basis. This approach will also be relevant for other rare diseases with significant genetic heterogeneity.

12.
Proc Natl Acad Sci U S A ; 112(15): E1898-907, 2015 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-25827233

RESUMO

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbß3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbß3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and ß3 C547G severely reduced αIIbß3 expression, whereas αIIb P943A partially reduced αIIbß3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69-98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or ß3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbß3 and highlight the challenges in predicting the clinical significance of novel missense variants.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação de Sentido Incorreto , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Trombastenia/genética , Alelos , Bases de Dados de Ácidos Nucleicos , Exoma/genética , Fibrinogênio/química , Fibrinogênio/metabolismo , Frequência do Gene , Células HEK293 , Humanos , Immunoblotting , Modelos Moleculares , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Trombastenia/metabolismo
13.
Stem Cells Dev ; 21(2): 181-90, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21504359

RESUMO

Amphibian oocytes have the special ability to directly induce the transcription of pluripotency and other genes in transplanted somatic nuclei. To this extent, oocytes induce a stem cell-like pattern of transcription in somatic cell nuclei. We ask whether the induced transcription in transplanted nuclei reflects the normal transcriptional activity of oocyte genes. We describe here the transcript content of a wide range of genes in Xenopus tropicalis oocytes. Using accurate quantitation, we find that each mature oocyte has accumulated several hundred transcripts of cell-type specific genes. This value is several orders of magnitude greater than the "leakage" level found in most somatic cells and about the same level found in somatic cells where these genes are fully expressed. Illumina sequencing confirms the high transcript content of a mature Xenopus oocyte. Most of the transcripts from these highly expressed genes in oocytes are correctly and efficiently spliced. Our results contribute a more quantitative view of certain amphibian oocyte transcripts than previously available. Our results also show that transplanted somatic nuclei conform, with respect to the genes analyzed, to the transcriptional characteristics of the recipient oocytes.


Assuntos
Núcleo Celular/genética , Técnicas de Transferência Nuclear , Oócitos , RNA Mensageiro/genética , Transcriptoma/genética , Proteínas de Xenopus/genética , Animais , Núcleo Celular/metabolismo , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Oócitos/citologia , Oócitos/metabolismo , Processamento de RNA , RNA Mensageiro/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Ativação Transcricional , Xenopus , Proteínas de Xenopus/metabolismo
14.
Circulation ; 124(22): 2411-22, 2011 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-22025602

RESUMO

BACKGROUND: The epigenome refers to marks on the genome, including DNA methylation and histone modifications, that regulate the expression of underlying genes. A consistent profile of gene expression changes in end-stage cardiomyopathy led us to hypothesize that distinct global patterns of the epigenome may also exist. METHODS AND RESULTS: We constructed genome-wide maps of DNA methylation and histone-3 lysine-36 trimethylation (H3K36me3) enrichment for cardiomyopathic and normal human hearts. More than 506 Mb sequences per library were generated by high-throughput sequencing, allowing us to assign methylation scores to ≈28 million CG dinucleotides in the human genome. DNA methylation was significantly different in promoter CpG islands, intragenic CpG islands, gene bodies, and H3K36me3-enriched regions of the genome. DNA methylation differences were present in promoters of upregulated genes but not downregulated genes. H3K36me3 enrichment itself was also significantly different in coding regions of the genome. Specifically, abundance of RNA transcripts encoded by the DUX4 locus correlated to differential DNA methylation and H3K36me3 enrichment. In vitro, Dux gene expression was responsive to a specific inhibitor of DNA methyltransferase, and Dux siRNA knockdown led to reduced cell viability. CONCLUSIONS: Distinct epigenomic patterns exist in important DNA elements of the cardiac genome in human end-stage cardiomyopathy. The epigenome may control the expression of local or distal genes with critical functions in myocardial stress response. If epigenomic patterns track with disease progression, assays for the epigenome may be useful for assessing prognosis in heart failure. Further studies are needed to determine whether and how the epigenome contributes to the development of cardiomyopathy.


Assuntos
Progressão da Doença , Epigenômica , Regulação da Expressão Gênica/fisiologia , Insuficiência Cardíaca/genética , Estudos de Casos e Controles , Ilhas de CpG/genética , Ilhas de CpG/fisiologia , Metilação de DNA/fisiologia , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/fisiopatologia , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Prognóstico
15.
Development ; 136(16): 2695-703, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19605498

RESUMO

Transplantation into eggs or oocytes is an effective means of achieving the reprogramming of somatic cell nuclei. We ask here whether the provision of gene-specific transcription factors forms part of the mechanism by which a gene that is repressed in somatic cells is transcribed in oocytes. We find that M1 oocytes have an extremely strong transcription-inducing activity. They cause muscle genes of nuclei from non-muscle somatic cells, after injection into oocytes, to be transcribed to nearly the same extent as muscle genes in muscle cells. We show, surprisingly, that the myogenic factor MyoD and other known myogenic factors are not required to induce the transcription of muscle genes in a range of non-muscle somatic cell nuclei after transplantation to Xenopus oocytes. The overexpression of Id, a dominant-negative repressor of MyoD, prevents maternal MyoD from binding to its consensus sequences; nevertheless, muscle genes are activated in somatic nuclei to the same extent as without Id. We conclude that M1 oocytes can reprogram somatic nuclei in a different way to other experimental procedures: oocytes do not suppress the transcription of inappropriate genes and they activate a gene without the help of its known transcription factors. We suggest that these characteristics might be a special property of amphibian oocytes, and possibly of oocytes in general.


Assuntos
Núcleo Celular/fisiologia , Músculos , Fatores de Regulação Miogênica/metabolismo , Técnicas de Transferência Nuclear , Oócitos , Transcrição Genética , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Músculos/citologia , Músculos/fisiologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fatores de Regulação Miogênica/genética , Oócitos/citologia , Oócitos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
16.
J Cell Sci ; 115(Pt 12): 2497-504, 2002 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12045220

RESUMO

Ryanodine receptors (RyRs) are expressed on the endoplasmic reticulum of many cells, where they form intracellular Ca2+-release channels that participate in the generation of intracellular Ca2+ signals. Here we report studies on the intracellular localisation and functional properties of transfected RyR1 or RyR3 channels in HEK 293 cells. Immunofluorescence studies indicated that both RyR1 and RyR3 did not form clusters but were homogeneously distributed throughout the endoplasmic reticulum. Ca2+ release experiments showed that transfected RyR1 and RyR3 channels responded to caffeine, although with different sensitivity, generating a global release of Ca2+ from the entire endoplasmic reticulum. However, video imaging and confocal microscopy analysis revealed that, in RyR3-expressing cells, local spontaneous Ca2+ release events were observed. No such spontaneous activity was observed in RyR1-expressing cells or in control cells. Interestingly, the spontaneous release events observed in RyR3-expressing cells were restricted to one or two regions of the endoplasmic reticulum, suggesting the formation of a further subcellular organisation of RyR3 in Ca2+ release units. These results demonstrate that different RyR isoforms can engage in the generation of distinct intracellular Ca2+ signals in HEK 293 cells.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células Eucarióticas/metabolismo , Líquido Intracelular/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Compartimento Celular/fisiologia , Células Cultivadas , Células Clonais/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Células Eucarióticas/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Líquido Intracelular/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Transfecção
17.
Biophys J ; 82(3): 1319-28, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11867448

RESUMO

Most adult mammalian skeletal muscles contain only one isoform of ryanodine receptor (RyR1), whereas neonatal muscles contain two isoforms (RyR1 and RyR3). Membrane depolarization fails to evoke calcium release in muscle cells lacking RyR1, demonstrating an essential role for this isoform in excitation-contraction coupling. In contrast, the role of RyR3 is unknown. We studied the participation of RyR3 in calcium release in wild type (containing both RyR1 and RyR3 isoforms) and RyR3-/- (containing only RyR1) myotubes in the presence or absence of imperatoxin A (IpTxa), a high-affinity agonist of ryanodine receptors. IpTxa significantly increased the amplitude and the rate of release only in wild-type myotubes. Calcium currents, recorded simultaneously with the transients, were not altered with IpTxa treatment. [(3)H]ryanodine binding to RyR1 or RyR3 was significantly increased in the presence of IpTxa. Additionally, IpTxa modified the gating and conductance level of single RyR1 or RyR3 channels when studied in lipid bilayers. Our data show that IpTxa can interact with both RyRs and that RyR3 is functional in myotubes and it can amplify the calcium release signal initiated by RyR1, perhaps through a calcium-induced mechanism. In addition, our data indicate that when RyR3-/- myotubes are voltage-clamped, the effect of IpTxa is not detected because RyR1s are under the control of the dihydropyridine receptor.


Assuntos
Cálcio/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Venenos de Escorpião/farmacologia , Animais , Animais Recém-Nascidos , Células CHO , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Cricetinae , Bicamadas Lipídicas/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Liso/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Isoformas de Proteínas , Rianodina/farmacologia , Fatores de Tempo
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