RESUMEN
Diagnosing mitochondrial disorders is a challenge due to the heterogeneous clinical presentation and large number of associated genes. A custom next generation sequencing (NGS) panel was developed incorporating the full mitochondrial genome (mtDNA) plus 19 nuclear genes involved in structural mitochondrial defects and mtDNA maintenance. This assay is capable of simultaneously detecting small gene sequence variations and larger copy number variants (CNVs) in both the nuclear and mitochondrial components along with heteroplasmy detection down to 5%. We describe technical validations of this panel and its implementation for clinical testing in a Canadian reference laboratory, and report its clinical performance in the initial 950 patients tested. Using this assay, we demonstrate a diagnostic yield of 18.1% of patients with known pathogenic variants. In addition to the common 5 kb mtDNA deletion, we describe significant contribution of pathogenic CNVs in both the mitochondrial genome and nuclear genes in this patient population.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Genética de Población , Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedades Mitocondriales/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Canadá/epidemiología , Núcleo Celular/genética , Niño , Preescolar , ADN Mitocondrial/genética , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mitocondrias/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/epidemiología , Enfermedades Mitocondriales/patología , Adulto JovenRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with increased risk for developing Parkinson's disease (PD). Previously, we found that LRRK2 G2019S mutation carriers have increased mitochondrial DNA (mtDNA) damage and after zinc finger nuclease-mediated gene mutation correction, mtDNA damage was no longer detectable. While the mtDNA damage phenotype can be unambiguously attributed to the LRRK2 G2019S mutation, the underlying mechanism(s) is unknown. Here, we examine the role of LRRK2 kinase function in LRRK2 G2019S-mediated mtDNA damage, using both genetic and pharmacological approaches in cultured neurons and PD patient-derived cells. Expression of LRRK2 G2019S induced mtDNA damage in primary rat midbrain neurons, but not in cortical neuronal cultures. In contrast, the expression of LRRK2 wild type or LRRK2 D1994A mutant (kinase dead) had no effect on mtDNA damage in either midbrain or cortical neuronal cultures. In addition, human LRRK2 G2019S patient-derived lymphoblastoid cell lines (LCL) demonstrated increased mtDNA damage relative to age-matched controls. Importantly, treatment of LRRK2 G2019S expressing midbrain neurons or patient-derived LRRK2 G2019S LCLs with the LRRK2 kinase inhibitor GNE-7915, either prevented or restored mtDNA damage to control levels. These findings support the hypothesis that LRRK2 G2019S-induced mtDNA damage is LRRK2 kinase activity dependent, uncovering a novel pathological role for this kinase. Blocking or reversing mtDNA damage via LRRK2 kinase inhibition or other therapeutic approaches may be useful to slow PD-associated pathology.
Asunto(s)
Daño del ADN , ADN Mitocondrial/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Enfermedad de Parkinson/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Células Cultivadas , ADN Mitocondrial/metabolismo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/patología , Persona de Mediana Edad , Mitocondrias/metabolismo , Morfolinas/farmacología , Mutación , Neuronas/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/enzimología , Polimorfismo de Nucleótido Simple , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Levels of the HER2/ErbB2 protein in the heart are upregulated in some women during breast cancer therapy, and these women are at high risk for developing heart dysfunction after sequential treatment with anti-ErbB2/trastuzumab or doxorubicin. Doxorubicin is known to increase oxidative stress in the heart, and thus we considered the possibility that ErbB2 protein influences the status of cardiac antioxidant defenses in cardiomyocytes. In this study, we measured reactive oxygen species (ROS) in cardiac mitochondria and whole hearts from mice with cardiac-specific overexpression of ErbB2 (ErbB2(tg)) and found that, compared with control mice, high levels of ErbB2 in myocardium result in lower levels of ROS in mitochondria (P = 0.0075) and whole hearts (P = 0.0381). Neonatal cardiomyocytes isolated from ErbB2(tg) hearts have lower ROS levels and less cellular death (P < 0.0001) following doxorubicin treatment. Analyzing antioxidant enzyme levels and activities, we found that ErbB2(tg) hearts have increased levels of glutathione peroxidase 1 (GPx1) protein (P < 0.0001) and GPx activity (P = 0.0031) in addition to increased levels of two known GPx activators, c-Abl (P = 0.0284) and Arg (P < 0.0001). Interestingly, although mitochondrial ROS emission is reduced in the ErbB2(tg) hearts, oxygen consumption rates and complex I activity are similar to control littermates. Compared with these in vivo studies, H9c2 cells transfected with ErbB2 showed less cellular toxicity and produced less ROS (P < 0.0001) after doxorubicin treatment but upregulated GR activity (P = 0.0237) instead of GPx. Our study shows that ErbB2-dependent signaling contributes to antioxidant defenses and suggests a novel mechanism by which anticancer therapies involving ErbB2 antagonists can harm myocardial structure and function.
Asunto(s)
Antineoplásicos/toxicidad , Antioxidantes/metabolismo , Doxorrubicina/toxicidad , Glutatión Peroxidasa/metabolismo , Cardiopatías/prevención & control , Mitocondrias Cardíacas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Receptor ErbB-2/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Glutatión Reductasa/metabolismo , Cardiopatías/inducido químicamente , Cardiopatías/enzimología , Cardiopatías/genética , Cardiopatías/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Ratas , Receptor ErbB-2/genética , Glutatión Peroxidasa GPX1RESUMEN
Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)-dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N-methyltransferase 2E-antisense 1 (KMT2E-AS1) and histone lysine N-methyltransferase 2E (KMT2E). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α-dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)-specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1/KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.
Asunto(s)
Hipertensión Pulmonar , ARN Largo no Codificante , Humanos , Ratas , Animales , Ratones , Alelos , Hipertensión Pulmonar/genética , Histonas , ARN Largo no Codificante/genética , Roedores , Lisina , Hipertensión Pulmonar Primaria Familiar , Hipoxia/genética , Metiltransferasas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genéticaRESUMEN
Petite Integration Factor 1 (PIF1) is a multifunctional helicase present in nuclei and mitochondria. PIF1 knock out (KO) mice exhibit accelerated weight gain and decreased wheel running on a normal chow diet. In the current study, we investigated whether Pif1 ablation alters whole body metabolism in response to weight gain. PIF1 KO and wild type (WT) C57BL/6J mice were fed a Western diet (WD) rich in fat and carbohydrates before evaluation of their metabolic phenotype. Compared with weight gain-resistant WT female mice, WD-fed PIF1 KO females, but not males, showed accelerated adipose deposition, decreased locomotor activity, and reduced whole-body energy expenditure without increased dietary intake. Surprisingly, PIF1 KO females did not show obesity-induced alterations in fasting blood glucose and glucose clearance. WD-fed PIF1 KO females developed mild hepatic steatosis and associated changes in liver gene expression that were absent in weight-matched, WD-fed female controls, linking hepatic steatosis to Pif1 ablation rather than increased body weight. WD-fed PIF1 KO females also showed decreased expression of inflammation-associated genes in adipose tissue. Collectively, these data separated weight gain from inflammation and impaired glucose homeostasis. They also support a role for Pif1 in weight gain resistance and liver metabolic dysregulation during nutrient stress.
Asunto(s)
ADN Helicasas/deficiencia , Dieta Occidental , Glucosa/metabolismo , Mediadores de Inflamación/metabolismo , Aumento de Peso/genética , Tejido Adiposo/metabolismo , Animales , Composición Corporal , Colesterol/metabolismo , Citocinas/metabolismo , Metabolismo Energético , Hígado Graso/metabolismo , Hígado Graso/patología , Femenino , Prueba de Tolerancia a la Glucosa , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Actividad MotoraRESUMEN
Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.
Asunto(s)
ADN Mitocondrial/genética , Enfermedades Mitocondriales/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Eliminación de Secuencia , Línea Celular , Humanos , Sensibilidad y EspecificidadRESUMEN
Mutations in genes coding for mitochondrial helicases such as TWINKLE and DNA2 are involved in mitochondrial myopathies with mtDNA instability in both human and mouse. We show that inactivation of Pif1, a third member of the mitochondrial helicase family, causes a similar phenotype in mouse. pif1-/- animals develop a mitochondrial myopathy with respiratory chain deficiency. Pif1 inactivation is responsible for a deficiency to repair oxidative stress-induced mtDNA damage in mouse embryonic fibroblasts that is improved by complementation with mitochondrial isoform mPif1(67). These results open new perspectives for the exploration of patients with mtDNA instability disorders.
Asunto(s)
ADN Helicasas/antagonistas & inhibidores , Silenciador del Gen , Miopatías Mitocondriales/genética , Animales , Células Cultivadas , Fibroblastos/fisiología , Ratones , Ratones Noqueados , Enfermedades MitocondrialesRESUMEN
AIMS: Despite the observation that ErbB2 regulates sensitivity of the heart to doxorubicin or ErbB2-targeted cancer therapies, mechanisms that regulate ErbB2 expression and activity have not been studied. Since isoproterenol up-regulates ErbB2 in kidney and salivary glands and ß2AR and ErbB2 complex in brain and heart, we hypothesized that ß-adrenergic receptors (AR) modulate ErbB2 signalling status. METHODS AND RESULTS: ErbB2 transfection of HEK293 cells up-regulates ß2AR, and ß2AR transfection of HEK293 up-regulates ErbB2. Interestingly, cardiomyocytes isolated from myocyte-specific ErbB2-overexpressing (ErbB2(tg)) mice have amplified response to selective ß2-agonist zinterol, and right ventricular trabeculae baseline force generation is markedly reduced with ß2-antagonist ICI-118 551. Consistently, receptor binding assays and western blotting demonstrate that ß2ARs levels are markedly increased in ErbB2(tg) myocardium and reduced by EGFR/ErbB2 inhibitor, lapatinib. Intriguingly, acute treatment of mice with ß1- and ß2-AR agonist isoproterenol resulted in myocardial ErbB2 increase, while inhibition with either ß1- or ß2-AR antagonist did not completely prevent isoproterenol-induced ErbB2 expression. Furthermore, inhibition of ErbB2 kinase predisposed mice hearts to injury from chronic isoproterenol treatment while significantly reducing isoproterenol-induced pAKT and pERK levels, suggesting ErbB2's role in transactivation in the heart. CONCLUSION: Our studies show that myocardial ErbB2 and ßAR signalling are linked in a feedback loop with ßAR activation leading to increased ErbB2 expression and activity, and increased ErbB2 activity regulating ß2AR expression. Most importantly, ErbB2 kinase activity is crucial for cardioprotection in the setting of ß-adrenergic stress, suggesting that this mechanism is important in the pathophysiology and treatment of cardiomyopathy induced by ErbB2-targeting antineoplastic drugs.
Asunto(s)
Isoproterenol/farmacología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , AMP Cíclico/metabolismo , Femenino , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ratones , Miocitos Cardíacos/efectos de los fármacos , Receptor ErbB-2/genética , Receptores Adrenérgicos beta 1/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored. METHODOLOGY/PRINCIPAL FINDINGS: We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2-3 times larger than those of control littermates, express increased atrial natriuretic peptide and ß-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib. CONCLUSIONS/SIGNIFICANCE: These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling pathways and induce a phenotype consistent with Hypertrophic Cardiomyopathy. Furthermore, our work suggests that in the situation where ErbB2 signaling contributes to cardiac hypertrophy, inhibition of this pathway may reverse this process.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Receptor ErbB-2/genética , Transducción de Señal/genética , Animales , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/genética , Cardiomiopatía Hipertrófica/fisiopatología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Fenómenos Electrofisiológicos , Expresión Génica , Isoproterenol/farmacología , Lapatinib , Masculino , Ratones , Ratones Transgénicos , Tamaño de los Órganos/efectos de los fármacos , Especificidad de Órganos , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Quinazolinas/farmacología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Antibodies recognizing the complement of the middle of PR3 (cPR3m) occur in â¼30% of PR3-anti-neutrophil cytoplasmic autoantibodies (ANCA)-vasculitis patients and immunization of animals with a peptide complementary to the middle of PR3 (cPR3m) induces not only anti-complementary PR3 antibodies, but also anti-PR3 antibodies derived through an anti-idiotypic response. PR3 epitopes recognized by patient ANCA, however, are not restricted to the middle of PR3. This prompted us to test for antibodies that react with proteins complementary to the terminal regions of PR3 (cPR3C and cPR3N) in PR3-ANCA patients. Anti-cPR3C reactivity was detected in 28% of patients but anti-cPR3N reactivity in only 15%. Ranked anti-cPR3C and anti-cPR3m reactivity correlated in the cohort, whereas there was no significant relationship between cPR3C and cPR3N reactivity. Serial samples from 3 patients' revealed that anti-cPR3C and anti-cPR3m reactivity followed a similar pattern over time. Serial samples from a fourth patient demonstrated an anti-cPR3N response without concurrent cPR3m or cPR3C reactivity. Epitope determination by mass spectrometry identified a 13-amino acid sequence on cPR3C that contained a common binding site recognized by antibodies from three patients. This peptide sequence contains a "PHQ" motif which was reported to be the basis for cross-reactivity of anti-cPR3m antibodies with plasminogen. Why these antibodies are detected in only â¼30% of the patients remains unclear. The data reveal that it is not due to lack of inclusion of flanking regions of complementary PR3 during screening. Instead, quite unexpectedly, the data demonstrate that patients' antibodies react with a restricted epitope that exists in both cPR3m and cPR3C.