RESUMO
Hypertrophic Cardiomyopathy (HCM) has been related to many different mutations in more than 20 different, mostly sarcomeric proteins. While development of the HCM-phenotype is thought to be triggered by the different mutations, a common mechanism remains elusive. Studying missense-mutations in the ventricular beta-myosin heavy chain (ß-MyHC, MYH7) we hypothesized that significant contractile heterogeneity exists among individual cardiomyocytes of HCM-patients that results from cell-to-cell variation in relative expression of mutated vs. wildtype ß-MyHC. To test this hypothesis, we measured force-calcium-relationships of cardiomyocytes isolated from myocardium of heterozygous HCM-patients with either ß-MyHC-mutation Arg723Gly or Arg200Val, and from healthy controls. From the myocardial samples of the HCM-patients we also obtained cryo-sections, and laser-microdissected single cardiomyocytes for quantification of mutated vs. wildtype MYH7-mRNA using a single cell RT-qPCR and restriction digest approach. We characterized gene transcription by visualizing active transcription sites by fluorescence in situ hybridization of intronic and exonic sequences of MYH7-pre-mRNA. For both mutations, cardiomyocytes showed large cell-to-cell variation in Ca++-sensitivity. Interestingly, some cardiomyocytes were essentially indistinguishable from controls what might indicate that they had no mutant ß-MyHC while others had highly reduced Ca++-sensitivity suggesting substantial fractions of mutant ß-MyHC. Single-cell MYH7-mRNA-quantification in cardiomyocytes of the same patients revealed high cell-to-cell variability of mutated vs. wildtype mRNA, ranging from essentially pure mutant to essentially pure wildtype MYH7-mRNA. We found 27% of nuclei without active transcription sites which is inconsistent with continuous gene transcription but suggests burst-like transcription of MYH7. Model simulations indicated that burst-like, stochastic on/off-switching of MYH7 transcription, which is independent for mutant and wildtype alleles, could generate the observed cell-to-cell variation in the fraction of mutant vs. wildtype MYH7-mRNA, a similar variation in ß-MyHC-protein, and highly heterogeneous Ca++-sensitivity of individual cardiomyocytes. In the long run, such contractile imbalance in the myocardium may well induce progressive structural distortions like cellular and myofibrillar disarray and interstitial fibrosis, as they are typically observed in HCM.
RESUMO
HCM, the most common inherited cardiac disease, is mainly caused by mutations in sarcomeric genes. More than a third of the patients are heterozygous for mutations in the MYH7 gene encoding for the ß-myosin heavy chain. In HCM-patients, expression of the mutant and the wildtype allele can be unequal, thus leading to fractions of mutant and wildtype mRNA and protein which deviate from 1:1. This so-called allelic imbalance was detected in whole tissue samples but also in individual cells. There is evidence that the severity of HCM not only depends on the functional effect of the mutation itself, but also on the fraction of mutant protein in the myocardial tissue. Allelic imbalance has been shown to occur in a broad range of genes. Therefore, we aimed to examine whether the MYH7-alleles are intrinsically expressed imbalanced or whether the allelic imbalance is solely associated with the disease. We compared the expression of MYH7-alleles in non-HCM donors and in HCM-patients with different MYH7-missense mutations. In the HCM-patients, we identified imbalanced as well as equal expression of both alleles. Also at the protein level, allelic imbalance was determined. Most interestingly, we also discovered allelic imbalance and balance in non-HCM donors. Our findings therefore strongly indicate that apart from mutation-specific mechanisms, also non-HCM associated allelic-mRNA expression regulation may account for the allelic imbalance of the MYH7 gene in HCM-patients. Since the relative amount of mutant mRNA and protein or the extent of allelic imbalance has been associated with the severity of HCM, individual analysis of the MYH7-allelic expression may provide valuable information for the prognosis of each patient.
Assuntos
Alelos , Desequilíbrio Alélico , Miosinas Cardíacas , Cardiomiopatia Hipertrófica , Regulação Enzimológica da Expressão Gênica , Cadeias Pesadas de Miosina , Sarcômeros , Adulto , Miosinas Cardíacas/biossíntese , Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/genética , Sarcômeros/genética , Sarcômeros/metabolismo , Sarcômeros/patologiaRESUMO
BACKGROUND: Mitral valve prolapse syndrome (MVPS) and MASS phenotype (MASS) are Marfan-like syndromes that exhibit aortic dilatation and mitral valve prolapse. Unlike in Marfan syndrome (MFS), the presence of ectopia lentis and aortic aneurysm preclude diagnosis of MVPS and MASS. However, it is unclear whether aortic dilatation and mitral valve prolapse remain stable in MVPS or MASS or whether they progress like in MFS. METHODS: This retrospective longitudinal observational study examines clinical characteristics and long-term prognosis of 44 adults with MVPS or MASS (18 men, 26 women aged 38 ± 17 years) as compared with 81 adults with Marfan syndrome (MFS) with similar age and sex distribution. The age at final contact was 42 ± 15 years with mean follow-up of 66 ± 49 months. RESULTS: At baseline, ectopia lentis and aortic sinus aneurysm were absent in MVPS and MASS, and systemic scores defined by the revised Ghent nosology were lower than in MFS (all P < .001). Unlike in MFS, no individual with MVPS and MASS developed aortic complications (P < .001). In contrast, the incidence of endocarditis (P = .292), heart failure (P = .644), and mitral valve surgery (P = .140) was similar in all syndromes. Cox regression analysis identified increased LV end-diastolic (P = .013), moderate MVR (P = .019) and flail MV leaflet (P = .017) as independent predictors of mitral valve surgery. CONCLUSIONS: The study provides evidence that MVPS and MASS are Marfan-like syndromes with stability of aortic dilatation but with progression of mitral valve prolapse. Echocardiographic characteristics of mitral valve disease rather than the type of syndrome, predict clinical progression of mitral valve prolapse.
Assuntos
Aneurisma da Aorta Torácica/diagnóstico , Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/diagnóstico , Dissecção Aórtica/genética , Testes Genéticos , Dissecção Aórtica/metabolismo , Dissecção Aórtica/fisiopatologia , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/fisiopatologia , Biomarcadores/metabolismo , Colágeno/genética , Colágeno/metabolismo , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Qualidade de Vida , Sensibilidade e Especificidade , Terminologia como Assunto , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by mutations in different structural genes and induces pathological hypertrophy with sudden cardiac death as a possible consequence. HCM can be separated into hypertrophic non-obstructive and obstructive cardiomyopathy (HNCM/HOCM) with different clinical treatment approaches. We here distinguished between HNCM, HOCM, cardiac amyloidosis and aortic stenosis by using microRNA profiling and investigated potential interactions between circulating miRNA levels and the most common mutations in MYH7and MYBPC3 genes. METHODS: Our study included 4 different groups: 23 patients with HNCM, 28 patients with HOCM, 47 patients with aortic stenosis and 22 healthy controls. Based on previous findings, 8 different cardiovascular known microRNAs (miR-1, miR-21, miR-29a, miR-29b, miR-29c, miR-133a, miR-155 and miR-499) were studied in serum of all patients and compared with clinically available patient data. RESULTS: We found miR-29a levels to be increased in patients with HOCM and correlating markers of cardiac hypertrophy. This was not the case in HNCM patients. In contrast, we identified miR-29c to be upregulated in aortic stenosis but not the other patient groups. ROC curve analysis of miR-29a/c distinguished between HOCM patients and aortic stenosis patients. MiR-29a and miR-155 levels discriminated HNCM patients from patients with senile cardiac amyloidosis. MiR-29a increased mainly in HOCM patients with a mutation in MYH7, whereas miR-155 was decreased in hypertrophic cardiomyopathy patients with a mutation in MYBPC3. CONCLUSION: We demonstrated that miR-29a and miR-29c show a specific signature to distinguish between aortic stenosis, hypertrophic non-obstructive and obstructive cardiomyopathies and thus could be developed into clinically useful biomarkers.
Assuntos
Estenose da Valva Aórtica/diagnóstico , Cardiomiopatia Hipertrófica/diagnóstico , MicroRNAs/sangue , Adulto , Idoso , Amiloidose/genética , Biomarcadores/sangue , Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/classificação , Proteínas de Transporte/genética , Diagnóstico Diferencial , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Cadeias Pesadas de Miosina/genética , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Echocardiographic upper normal limits of both main pulmonary artery (MPA) diameters (MPA-d) and ratio of MPA to aortic root diameter (MPA-r) are not defined in healthy adults. Accordingly, frequency of MPA dilatation based on echocardiography remains to be assessed in adults with Marfan syndrome (MFS). METHODS: We enrolled 123 normal adults (72 men, 52 women aged 42 ± 14 years) and 98 patients with MFS (42 men, 56 women aged 39 ± 14 years) in a retrospective cross-sectional observational controlled study in four tertiary care centers. We defined outcome measures including upper normal limits of MPA-d and MPA-r as 95 quantile of normal persons, MPA dilatation as diameters > upper normal limits, MPA aneurysm as diameters >4 cm, and indication for surgery as MPA diameters >6 cm. RESULTS: MPA diameters revealed normal distribution without correlation to age, sex, body weight, body height, body mass index and body surface area. The upper normal limit was 2.6 cm (95% confidence interval (CI) =2.44-2.76 cm) for MPA-d, and 1.05 (95% CI = .86-1.24) for MPA-r. MPA dilatation presented in 6 normal persons (4.9%) and in 68 MFS patients (69.4%; P < .001), MPA aneurysm presented only in MFS (15 patients; 15.3%; P < .001), and no patient required surgery. Mean MPA-r were increased in MFS (P < .001), but ratios >1.05 were equally frequent in 7 normal persons (5%) and in 8 MFS patients (10.5%; P = .161). MPA-r related to aortic root diameters (P = .042), reduced left ventricular ejection fraction (P = .006), and increased pulmonary artery systolic pressures (P = .040). No clinical manifestations of MFS and no FBN1 mutation characteristics related to MPA diameters. CONCLUSIONS: We established 2.6 cm for MPA-d and 1.05 for MPA-r as upper normal limits. MFS exhibits a high prevalence of MPA dilatation and aneurysm. However, patients may require MPA surgery only in scarce circumstances, most likely because formation of marked MPA aneurysm may require LV dysfunction and increased PASP.
Assuntos
Aneurisma Aórtico/diagnóstico por imagem , Síndrome de Marfan/diagnóstico por imagem , Artéria Pulmonar/diagnóstico por imagem , Vasodilatação , Adolescente , Adulto , Idoso , Aneurisma Aórtico/fisiopatologia , Estudos Transversais , Ecocardiografia/normas , Feminino , Humanos , Masculino , Síndrome de Marfan/fisiopatologia , Pessoa de Meia-Idade , Artéria Pulmonar/fisiopatologia , Valores de Referência , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUNDS: The Loeys-Dietz syndrome (LDS) is an inherited connective tissue disorder caused by mutations in the transforming growth factor ß (TGF-ß) receptors TGFBR1 or TGFBR2. Most patients with LDS develop severe aortic aneurysms resulting in early need of surgical intervention. In order to gain further insight into the pathophysiology of the disorder, we investigated circulating outgrowth endothelial cells (OEC) from the peripheral blood of LDS patients from a cohort of 23 patients including 6 patients with novel TGF-ß receptor mutations. METHODS AND RESULTS: We performed gene expression profiling of OECs using microarray analysis followed by quantitative PCR for verification of gene expression. Compared to OECs of age- and sex-matched healthy controls, OECs isolated from three LDS patients displayed altered expression of several genes belonging to the TGF-ß pathway, especially those affecting bone morphogenic protein (BMP) signalling including BMP2, BMP4 and BMPR1A. Gene expression of BMP antagonist Gremlin-1 (GREM1) showed the most prominent up-regulation. This increase was confirmed at the protein level by immunoblotting of LDS-OECs. In immunohistochemistry, abundant Gremlin-1 protein expression could be verified in endothelial cells as well as smooth muscle cells within the arterial media. Furthermore, Gremlin-1 plasma levels of LDS patients were significantly elevated compared to healthy control subjects. CONCLUSIONS: These findings open new avenues in the understanding of the pathogenesis of Loeys-Dietz syndrome and the development of new diagnostic serological methods for early disease detection.
Assuntos
Células Endoteliais/patologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Síndrome de Loeys-Dietz/fisiopatologia , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Adulto , Aorta/metabolismo , Aorta/patologia , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 4/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/biossíntese , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Proliferação de Células/genética , Diagnóstico Precoce , Feminino , Perfilação da Expressão Gênica , Humanos , Proteínas de Ligação a TGF-beta Latente/biossíntese , Síndrome de Loeys-Dietz/diagnóstico , Síndrome de Loeys-Dietz/genética , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , RNA Mensageiro/biossíntese , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Adulto JovemRESUMO
BACKGROUND: Total serum transforming growth factor-beta 1 (tsTGF-ß1) is increased in patients with Marfan syndrome (MFS), but it has not been assessed in thoracic aortic aneurysm and dissection (TAAD), Loeys-Dietz syndrome (LDS), and bicuspid aortic valve disease (BAVD). HYPOTHESIS: tsTGF-ß1 is increased in genetic aortic syndromes including TAAD, LDS, MFS, and BAVD. METHODS: We measured tsTGF-ß1 and performed sequencing of the genes FBN1, TGFBR1, and TGFBR2 in 317 consecutive patients with suspected or known genetic aortic syndrome (167 men, 150 women; mean age 43 ± 14 years). TAAD was diagnosed in 20, LDS in 20, MFS in 128, and BAVD in 30 patients, and genetic aortic syndrome was excluded in 119 patients. RESULTS: Elevated tsTGF-ß1 levels were associated with causative gene mutations (P = 0.008), genetic aortic syndrome (P = 0.009), and sporadic occurrence of genetic aortic syndrome (P = 0.048), whereas only genetic aortic syndrome qualified as an independent predictor of tsTGF-ß1 (P = 0.001). The tsTGF-ß1 levels were elevated in FBN1 and NOTCH1 mutations vs patients without mutations (both P = 0.004), and in NOTCH1 mutations vs ACTA2/MYH11 mutations (P = 0.015). Similarly, tsTGF-ß1 levels were elevated in MFS (P = 0.003) and in BAVD (P = 0.006) vs patients without genetic aortic syndrome. In contrast to specific clinical features of MFS, FBN1 in-frame mutations (P = 0.019) were associated with increased tsTGF-ß1 levels. CONCLUSIONS: tsTGF-ß1 is elevated in the entire spectrum of genetic aortic syndromes. However, gradual differences in the increases of tsTGF-ß1 levels may mirror different degrees of alteration of tsTGF-ß1 signaling in different genetic aortic syndromes.
Assuntos
Aneurisma da Aorta Torácica/sangue , Valva Aórtica/anormalidades , Doenças das Valvas Cardíacas/sangue , Síndrome de Loeys-Dietz/sangue , Síndrome de Marfan/sangue , Fator de Crescimento Transformador beta1/sangue , Adolescente , Adulto , Idoso , Aneurisma da Aorta Torácica/genética , Doença da Válvula Aórtica Bicúspide , Feminino , Fibrilina-1 , Fibrilinas , Doenças das Valvas Cardíacas/genética , Humanos , Síndrome de Loeys-Dietz/genética , Masculino , Síndrome de Marfan/genética , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Mutação , Receptor Notch1/genética , Adulto JovemRESUMO
BACKGROUND: Marfan syndrome is associated with ventricular arrhythmia but risk factors including FBN1 mutation characteristics require elucidation. METHODS AND RESULTS: We performed an observational cohort study of 80 consecutive adults (30 men, 50 women aged 42±15 years) with Marfan syndrome caused by FBN1 mutations. We assessed ventricular arrhythmia on baseline ambulatory electrocardiography as >10 premature ventricular complexes per hour (>10 PVC/h), as ventricular couplets (Couplet), or as non-sustained ventricular tachycardia (nsVT), and during 31±18 months of follow-up as ventricular tachycardia (VT) events (VTE) such as sudden cardiac death (SCD), and sustained ventricular tachycardia (sVT). We identified >10 PVC/h in 28 (35%), Couplet/nsVT in 32 (40%), and VTE in 6 patients (8%), including 3 with SCD (4%). PVC>10/h, Couplet/nsVT, and VTE exhibited increased N-terminal pro-brain natriuretic peptide serum levels(P<.001). All arrhythmias related to increased NT-proBNP (P<.001), where PVC>10/h and Couplet/nsVT also related to increased indexed end-systolic LV diameters (Pâ=â.024 and Pâ=â.020), to moderate mitral valve regurgitation (Pâ=â.018 and Pâ=â.003), and to prolonged QTc intervals (Pâ=â.001 and Pâ=â.006), respectively. Moreover, VTE related to mutations in exons 24-32 (Pâ=â.021). Kaplan-Meier analysis corroborated an association of VTE with increased NT-proBNP (P<.001) and with mutations in exons 24-32 (P<.001). CONCLUSIONS: Marfan syndrome with causative FBN1 mutations is associated with an increased risk for arrhythmia, and affected persons may require life-long monitoring. Ventricular arrhythmia on electrocardiography, signs of myocardial dysfunction and mutations in exons 24-32 may be risk factors of VTE.
Assuntos
Arritmias Cardíacas/genética , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patologia , Proteínas dos Microfilamentos/genética , Adulto , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Morte Súbita Cardíaca/patologia , Eletrocardiografia Ambulatorial , Éxons/genética , Feminino , Fibrilina-1 , Fibrilinas , Humanos , Estimativa de Kaplan-Meier , Masculino , Síndrome de Marfan/genética , Pessoa de Meia-Idade , Mutação , Peptídeo Natriurético Encefálico/genética , Fragmentos de Peptídeos/genética , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologiaRESUMO
Thoracic aortic aneurysm and dissection is associated with increasing mortality rate that may occur as part of a syndrome or as an isolated familial condition. Several genes have been implicated in causing TAAD, though an appropriate genetic test for their parallel testing is not yet available. Herein, we describe the novel 117-kb "MFSTAAD chip" that may help to understand the genetic basis of TAAD. A custom duplicate resequencing assay was developed to cover eight genes previously described in TAAD; FBN1, TGFBR1&2, COL3A1, MYH11, ACTA2, SLC2A10 and NOTCH1. GSEQ and SeqC software were used for data analysis. The analytical sensitivity of the assay was validated by the recognition of 182 known mutations (153 point mutations, 21 deletions, 7 insertions and 1 duplication) and a cohort of 28 patients were selected to determine the mutation yield, whereby 18 of them were previously negative for mutations in the genes FBN1 and TGFBR2. The assay had significantly higher sensitivity for point mutations (100%) and the largest deletion of 16 bp was detectable through a decline in the hybridization strength. The overall analytical sensitivity was 85%. Mutation testing of 28 unrelated TAAD patients revealed 4 known and 6 possibly pathogenic mutations with a mutation yield of 32%. The MFSTAAD chip is an alternative tool to next-generation sequencing that allows parallel analysis of several genes on a single platform. Refinements in the probe design and data analysis software will increase the analytical sensitivity of insertions and deletions making this assay even more applicable for clinical testing.
Assuntos
Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/genética , Análise Mutacional de DNA/métodos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sequência de Bases , Simulação por Computador , Análise Mutacional de DNA/instrumentação , Reações Falso-Positivas , Fibrilina-1 , Fibrilinas , Predisposição Genética para Doença , Humanos , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Oligonucleotídeos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Until today, FBN1 gene mutation characteristics were not compared with clinical features for the prediction of mitral valve disease progression. METHODS: Therefore, we conducted a study of 116 patients (53 men, 63 women aged 33 ± 15 years) with a causative FBN1 gene mutation and ≤ moderate mitral valve regurgitation at baseline. RESULTS: During 7.4 ± 6.8 years 30 patients developed progression of mitral valve regurgitation ≥ 1 grade (primary endpoint), and 26 patients required mitral valve surgery (secondary endpoint). Cox regression analysis identified an association of atrial fibrillation (hazard ratio (HR)=2.703; 95% confidence interval (CI) 1.013-7.211; P=.047), left ventricular ejection fraction (HR=.970; 95%CI .944-.997; P=.032), indexed end-diastolic left ventricular diameter (HR=15.165; 95%CI 4.498-51.128; P<.001), indexed left atrial diameter (HR=1.107; 95%CI 1.045-1.173; P=.001), tricuspid valve prolapse (HR=2.599; 95%CI 1.243-5.437; P=.011), posterior leaflet prolapse (HR=1.075; 95%CI 1.023-1.130; P=.009), and posterior leaflet thickening (HR=3.368; 95%CI 1.265-8.968; P=.015) with progression of mitral valve disease, whereas none of the FBN1 gene mutation characteristics were associated with progression of mitral valve disease. However, Cox regression analysis identified a marginal relationship of FBN1 gene mutations located both in a transforming-growth-factor beta-binding protein-like (TGFb-BP) domain (HR=3.453; 95%CI .982-12.143; P=.053), and in the calcium-binding epidermal growth factor-like (cbEGF) domain (HR=2.909; 95%CI .957-8.848; P=.060) with mitral valve surgery, a finding that was corroborated by Kaplan-Meier analysis (P=.014; and P=.041, respectively). CONCLUSION: Clinical features were better predictors of mitral valve disease progression than FBN1 gene mutation characteristics.
Assuntos
Progressão da Doença , Proteínas dos Microfilamentos/genética , Insuficiência da Valva Mitral/diagnóstico , Insuficiência da Valva Mitral/genética , Mutação/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Fibrilina-1 , Fibrilinas , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Insuficiência da Valva Mitral/patologia , Adulto JovemRESUMO
In recent years, several new white matter diseases have been identified based on magnetic resonance imaging and clinical findings. For most newly defined disorders the genetic basis has been identified. However, there is still a large group of patients without a specific diagnosis. Hypomyelinating leukodystrophies are the largest group among them. In some disorders characterized by hypomyelination only central nervous system involvement is observed, but in some disorders involvement of other organs is observed as well, such as eyes or teeth. Pelizaeus-Merzbacher-like disease (PMLD) is an autosomal recessive hypomyelinating disorder of the central nervous system characterized by nystagmus, ataxia, and progressive spasticity. The disease is caused by mutations in GJC2, the gene that encodes the gap junction protein connexin 47. Here we describe hypomyelination and Müllerian agenesis syndrome in a girl who is homozygous for a novel mutation in the GJC2 gene. It is an open question whether this is an association by chance or a feature of PMLD not previously noted.
Assuntos
Encéfalo/patologia , Conexinas/genética , Doenças Desmielinizantes/genética , Ductos Paramesonéfricos/anormalidades , Mutação , Doença de Pelizaeus-Merzbacher/genética , Pelve/anormalidades , Adolescente , Doenças Desmielinizantes/patologia , Feminino , Humanos , Doença de Pelizaeus-Merzbacher/patologia , SíndromeRESUMO
Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 (FBN1) gene, and mutations in FBN1 are known to be responsible for over 90% of all MFS cases. Locus heterogeneity has also been reported and confirmed, with mutations in the receptor genes TGFBR1 and TGFBR2 identified in association with MFS-related phenotypes. It is now known that dysregulation of TGF-ß signaling is involved in MFS pathogenesis. To test the hypothesis that dysregulation of TGFBR3-associated TGF-ß signaling is implicated in MFS or related phenotype pathogenesis, we selected a cohort of 49 patients, fulfilling or nearly fulfilling the diagnostic criteria for MFS. The patients were known not to carry a mutation in the FBN1 gene (including three 5' upstream alternatively spliced exons), the TGFBR1 and TGFBR2 genes. Mutation screening for the TGFBR3 gene in these patients and in controls led to the identification of a total of ten exonic (one novel), four intronic (one novel) and one 3'UTR variant in the TGFBR3 gene. Our data suggest that variations in TGFBR3 gene appear not to be associated with MFS or related phenotype.
Assuntos
Predisposição Genética para Doença , Síndrome de Loeys-Dietz/genética , Síndrome de Marfan/genética , Polimorfismo de Nucleotídeo Único/genética , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Adolescente , Sequência de Aminoácidos , Feminino , Frequência do Gene/genética , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Proteoglicanas/química , Receptores de Fatores de Crescimento Transformadores beta/químicaRESUMO
Herpes simplex virus (HSV) immediate-early protein ICP0 is a transcriptional activator with E3 ubiquitin ligase activity that induces the degradation of ND10 proteins, including the promyelocytic leukemia protein (PML) and Sp100. Moreover, ICP0 has a role in the derepression of viral genomes and in the modulation of the host interferon response to virus infection. Here, we report that ICP0 interacts with SIAH-1, a cellular E3 ubiquitin ligase that is involved in multiple cellular pathways and is itself capable of mediating PML degradation. This novel virus-host interaction profoundly stabilized SIAH-1 and recruited this cellular E3 ligase into ICP0-containing nuclear bodies. Moreover, SIAH-1 mediated the polyubiquitination of HSV ICP0 in vitro and in vivo. After infection of SIAH-1 knockdown cells with HSV, higher levels of ICP0 were produced, ICP0 was less ubiquitinated, and the half-life of this multifunctional viral regulatory protein was increased. These results indicate an inhibitory role of SIAH-1 during lytic infection by targeting ICP0 for proteasomal degradation.
Assuntos
Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Interações Hospedeiro-Patógeno , Humanos , Hidrólise , Reação em Cadeia da Polimerase , Ligação Proteica , UbiquitinaçãoRESUMO
The inherited neurodegenerative disorder glutaric aciduria type 1 (GA1) results from mutations in the gene for the mitochondrial matrix enzyme glutaryl-CoA dehydrogenase (GCDH), which leads to elevations of the dicarboxylates glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in brain and blood. The characteristic clinical presentation of GA1 is a sudden onset of dystonia during catabolic situations, resulting from acute striatal injury. The underlying mechanisms are poorly understood, but the high levels of GA and 3OHGA that accumulate during catabolic illnesses are believed to play a primary role. Both GA and 3OHGA are known to be substrates for Na(+)-coupled dicarboxylate transporters, which are required for the anaplerotic transfer of the tricarboxylic acid cycle (TCA) intermediate succinate between astrocytes and neurons. We hypothesized that GA and 3OHGA inhibit the transfer of succinate from astrocytes to neurons, leading to reduced TCA cycle activity and cellular injury. Here, we show that both GA and 3OHGA inhibit the uptake of [(14)C]succinate by Na(+)-coupled dicarboxylate transporters in cultured astrocytic and neuronal cells of wild-type and Gcdh(-/-) mice. In addition, we demonstrate that the efflux of [(14)C]succinate from Gcdh(-/-) astrocytic cells mediated by a not yet identified transporter is strongly reduced. This is the first experimental evidence that GA and 3OHGA interfere with two essential anaplerotic transport processes: astrocytic efflux and neuronal uptake of TCA cycle intermediates, which occur between neurons and astrocytes. These results suggest that elevated levels of GA and 3OHGA may lead to neuronal injury and cell death via disruption of TCA cycle activity.
Assuntos
Astrócitos/metabolismo , Glutaratos/metabolismo , Neurônios/metabolismo , Ácido Succínico/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Transporte Biológico/genética , Encéfalo/metabolismo , Encefalopatias Metabólicas/genética , Encefalopatias Metabólicas/metabolismo , Morte Celular/genética , Linhagem Celular Transformada , Ciclo do Ácido Cítrico/genética , Glutaril-CoA Desidrogenase/deficiência , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Camundongos , Camundongos Knockout , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismoRESUMO
Glutaric aciduria type 1 (GA1) is an autosomal recessive neurometabolic disorder caused by mutations in the glutaryl-CoA dehydrogenase gene (GCDH), leading to an accumulation and high excretion of glutaric acid and 3-hydroxyglutaric acid. Considerable variation in severity of the clinical phenotype is observed with no correlation to the genotype. We report here for the first time on expression studies of four missense mutations c.412A > G (p.Arg138Gly), c.787A > G (p.Met263Val), c.1204C > T (p.Arg402Trp) and c.1240G > A (p.Glu414Lys) identified in GA1 patients in mammalian cells. Biochemical analyses revealed that all mutants were enzymatically inactive with the exception of p.Met263Val which showed 10% activity of the expressed wild-type enzyme. Western blot and pulse-chase analyses demonstrated that the amount of expressed p.Arg402Trp protein was significantly reduced compared with cells expressing wild-type protein which was due to rapid intramitochondrial degradation. Upon cross-linkage the formation of homotetrameric GCDH was strongly impaired in p.Met263Val and p.Arg402Trp mutants. In addition, GCDH appears to interact with distinct heterologous polypeptides to form novel 97, 130 and 200 kDa GCDH complexes. Molecular modeling of mutant GCDH suggests that Met263 at the surface of the GCDH protein might be part of the contact interface to interacting proteins. These results indicate that reduced intramitochondrial stability as well as the impaired formation of homo- and heteromeric GCDH complexes can underlie GA1.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Glutaratos/metabolismo , Glutaril-CoA Desidrogenase/química , Glutaril-CoA Desidrogenase/genética , Mutação de Sentido Incorreto/genética , Estrutura Quaternária de Proteína/genética , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Substituição de Aminoácidos/genética , Animais , Catálise , Linhagem Celular , Cricetinae , Ativação Enzimática/genética , Estabilidade Enzimática/genética , Regulação da Expressão Gênica/genética , Genes Recessivos , Glutaril-CoA Desidrogenase/metabolismo , Humanos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
Glutaric aciduria type 1 (GA1) is caused by the deficiency of glutaryl-CoA dehydrogenase (GCDH). Affected patients are prone to the development of encephalopathic crises during an early time window with destruction of striatal neurons and a subsequent irreversible movement disorder. 3-Hydroxyglutaric acid (3OHGA) accumulates in tissues and body fluids of GA1 patients and has been shown to mediate toxic effects on neuronal as well as endothelial cells. Injection of (3H)-labeled into 6 week-old Gcdh(-/-) mice, a model of GA1, revealed a low recovery in kidney, liver, or brain tissue that did not differ from control mice. Significant amounts of 3OHGA were found to be excreted via the intestinal tract. Exposure of Gcdh(-/-) mice to a high protein diet led to an encephalopathic crisis, vacuolization in the brain, and death after 4-5 days. Under these conditions, high amounts of injected 3H-3OHGA were found in kidneys of Gcdh(-/-) mice, whereas the radioactivity recovered in brain and blood was reduced. The data demonstrate that under conditions mimicking encephalopathic crises the blood-brain barrier appears to remain intact.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Encefalopatias/metabolismo , Glutaratos/metabolismo , Glutaril-CoA Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Animais , Transporte Biológico , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatias/etiologia , Dextranos/metabolismo , Modelos Animais de Doenças , Glutaril-CoA Desidrogenase/genética , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Patients with glutaryl-CoA dehydrogenase (GCDH) deficiency accumulate glutaric acid (GA) and 3-hydroxyglutaric acid (3OH-GA) in their blood and urine. To identify the transporter mediating the translocation of 3OH-GA through membranes, kidney tissue of Gcdh-/- mice have been investigated because of its central role in urinary excretion of this metabolite. Using microarray analyses of kidney-expressed genes in Gcdh-/- mice, several differentially expressed genes encoding transporter proteins were identified. Real-time polymerase chain reaction analysis confirmed the upregulation of the sodium-dependent dicarboxylate cotransporter 3 (NaDC3) and the organic cation transporter 2 (OCT2). Expression analysis of NaDC3 in Xenopus laevis oocytes by the two-electrode-voltage-clamp technique demonstrated the sodium-dependent translocation of 3OH-GA with a K (M) value of 0.95 mM. Furthermore, tracer flux measurements in Chinese hamster ovary cells overexpressing OCT2 showed that 3OH-GA inhibited significantly the uptake of methyl-4-phenylpyridinium, whereas 3OH-GA is not transported by OCT2. The data demonstrate for the first time the membrane translocation of 3OH-GA mediated by NaDC3 and the cis-inhibitory effect on OCT2-mediated transport of cations.