RESUMEN
For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.
Asunto(s)
Perfilación de la Expresión Génica , Trastornos del Neurodesarrollo , Humanos , RNA-Seq , Cicloheximida , Análisis de Secuencia de ARN/métodos , Trastornos del Neurodesarrollo/diagnóstico , Trastornos del Neurodesarrollo/genéticaRESUMEN
Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA-seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease-causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin-fibroblast-based transcriptome analysis for molecular diagnostics. RNA-seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA-based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant.
Asunto(s)
Neurofibromatosis 1 , Humanos , Neurofibromatosis 1/diagnóstico , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Mutación , Empalme del ARN/genética , ADN , Fibroblastos/patología , Neurofibromina 1/genéticaRESUMEN
Alternative splicing (AS) is crucial for cell-type-specific gene transcription and plays a critical role in neuronal differentiation and synaptic plasticity. De novo frameshift variants in NOVA2, encoding a neuron-specific key splicing factor, have been recently associated with a new neurodevelopmental disorder (NDD) with hypotonia, neurological features, and brain abnormalities. We investigated eight unrelated individuals by exome sequencing (ES) and identified seven novel pathogenic NOVA2 variants, including two with a novel localization at the KH1 and KH3 domains. In addition to a severe NDD phenotype, novel clinical features included psychomotor regression, attention deficit-hyperactivity disorder (ADHD), dyspraxia, and urogenital and endocrinological manifestations. To test the effect of the variants on splicing regulation, we transfected HeLa cells with wildtype and mutant NOVA2 complementary DNA (cDNA). The novel variants NM_002516.4:c.754_756delCTGinsTT p.(Leu252Phefs*144) and c.1329dup p.(Lys444Glnfs*82) all negatively affected AS events. The distal p.(Lys444Glnfs*82) variant, causing a partial removal of the KH3 domain, had a milder functional effect leading to an intermediate phenotype. Our findings expand the molecular and phenotypic spectrum of NOVA2-related NDD, supporting the pathogenic role of AS disruption by truncating variants and suggesting that this is a heterogeneous condition with variable clinical course.
Asunto(s)
Discapacidad Intelectual , Trastornos del Neurodesarrollo , Empalme Alternativo , Células HeLa , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Hipotonía Muscular/genética , Proteínas del Tejido Nervioso/genética , Antígeno Ventral Neuro-Oncológico , Trastornos del Neurodesarrollo/genética , Fenotipo , Proteínas de Unión al ARN/genéticaRESUMEN
Terminal osseous dysplasia with pigmentary defects (TODPD), also known as digitocutaneous dysplasia, is one of the X-linked filaminopathies caused by a variety of FLNA-variants. TODPD is characterized by skeletal defects, skin fibromata and dysmorphic facial features. So far, only a single recurrent variant (c.5217G>A;p.Val1724_Thr1739del) in FLNA has found to be responsible for TODPD. We identified a novel c.5217+5G>C variant in FLNA in a female proband with skeletal defects, skin fibromata, interstitial lung disease, epilepsy, and restrictive cardiomyopathy. This variant causes mis-splicing of exon 31 predicting the production of a FLNA-protein with an in-frame-deletion of 16 residues identical to the miss-splicing-effect of the recurrent TODPD c.5217G>A variant. This mis-spliced transcript was explicitly detected in heart tissue, but was absent from blood, skin, and lung. X-inactivation analyses showed extreme skewing with almost complete inactivation of the mutated allele (>90%) in these tissues, except for heart. The mother of the proband, who also has fibromata and skeletal abnormalities, is also carrier of the FLNA-variant and was diagnosed with noncompaction cardiomyopathy after cardiac screening. No other relevant variants in cardiomyopathy-related genes were found. Here we describe a novel variant in FLNA (c.5217+5G>C) as the second pathogenic variant responsible for TODPD. Cardiomyopathy has not been described as a phenotypic feature of TODPD before.
Asunto(s)
Cardiomiopatías/genética , Filaminas/genética , Dedos/anomalías , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Predisposición Genética a la Enfermedad , Deformidades Congénitas de las Extremidades/genética , Osteocondrodisplasias/genética , Trastornos de la Pigmentación/genética , Dedos del Pie/anomalías , Cardiomiopatías/complicaciones , Cardiomiopatías/patología , Preescolar , Femenino , Dedos/patología , Genes Ligados a X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/complicaciones , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Humanos , Lactante , Deformidades Congénitas de las Extremidades/complicaciones , Deformidades Congénitas de las Extremidades/patología , Mutación/genética , Osteocondrodisplasias/complicaciones , Osteocondrodisplasias/patología , Fenotipo , Trastornos de la Pigmentación/complicaciones , Trastornos de la Pigmentación/patología , Eliminación de Secuencia/genética , Dedos del Pie/patología , Inactivación del Cromosoma X/genéticaRESUMEN
Each year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next-generation sequencing (NGS). This requires pathogenicity classification of millions of DNA variants on the standard 5-tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch labs decided to publicly share their classifications. Variant classifications of nearly 100,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center were labeled as "consensus" when classifications agreed, and shared internationally with LOVD and ClinVar. When classifications opposed (LB/B vs. LP/P), they were labeled "conflicting", while other nonconsensus observations were labeled "no consensus". We assessed our classifications using the InterVar software to compare to ACMG 2015 guidelines, showing 99.7% overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5-tier classification system. The data sharing boosted the quality of DNA diagnostics in Dutch labs, an initiative we hope will be followed internationally. Recently, a positive match with a case from outside our consortium resulted in a more definite disease diagnosis.
Asunto(s)
Enfermedades Genéticas Congénitas/diagnóstico , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Difusión de la Información/métodos , Exactitud de los Datos , Bases de Datos Genéticas , Enfermedades Genéticas Congénitas/genética , Guías como Asunto , Humanos , Laboratorios , Países Bajos , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: Non-invasive prenatal testing (NIPT) detects placental chromosome aberrations. When amniocentesis reveals a normal karyotype, confined placental mosaicism (CPM) may be assumed. In order to confirm this, placental cytogenetic studies were performed. METHOD: NIPT was conducted in the course of the Dutch TRIDENT study. Placentas of 10 cases with NIPT results indicating an autosomal trisomy and showing a normal (N = 9) or low mosaic karyotype (N = 1) in amniotic fluid (AF) were investigated. The cytotrophoblast as well as the mesenchymal core of two to four placental chorionic villi biopsies were studied with single nucleotide polymorphism (SNP) array. Clinical outcome data were collected. RESULTS: In 10/10 cases, CPM was proven. In 3/10 cases trisomy/uniparental disomy (UPD)/biparental disomy (BPD) mosaicism was discovered. In 2/3 cases, all three cell lines were present in the placenta, whereas BPD was found in AF. In 1/3 cases trisomy 22/UPD22 was present in AF while trisomy 22/BPD22 mosaicism was found in the placenta. Five of 10 pregnancies were affected with pre-eclampsia, low birth weight, preterm delivery, and/or congenital malformations. CONCLUSION: The presence of trisomy/UPD/BPD mosaicism in 3/10 cases that we investigated proves that trisomic zygote rescue may involve multiple rescue events during early embryogenesis. UPD mosaicism, when present in crucial fetal tissues, may explain the abnormal phenotype in undiagnosed cases.
Asunto(s)
Mosaicismo , Enfermedades Placentarias/genética , Placenta/fisiopatología , Diagnóstico Prenatal/métodos , Trisomía/diagnóstico , Trisomía/genética , Disomía Uniparental/genética , Amniocentesis , Líquido Amniótico/fisiología , Femenino , Pruebas Genéticas , Humanos , Cariotipificación , Polimorfismo de Nucleótido Simple , Embarazo , Cigoto/fisiologíaRESUMEN
Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss-of-function variants in desmosomal genes leads to a variety of skin- and heart-related phenotypes. In this study, we report TUFT1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair, and mild palmoplantar keratoderma but without a cardiac phenotype, we identified a homozygous splice-site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of TUFT1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that TUFT1 is positioned within the desmosome and that its location is dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1-knockout mouse model mimicked the patients' phenotypes. Altogether, this study reveals TUFT1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair, and palmoplantar keratoderma.
Asunto(s)
Enfermedades del Cabello , Queratodermia Palmoplantar , Anomalías Cutáneas , Animales , Humanos , Ratones , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Desmosomas/metabolismo , Cabello/metabolismo , Enfermedades del Cabello/genética , Enfermedades del Cabello/metabolismo , Queratodermia Palmoplantar/genética , Queratodermia Palmoplantar/metabolismo , Piel/metabolismo , Anomalías Cutáneas/metabolismoRESUMEN
The aim of this article is to describe neurovascular findings in patients with Loeys Dietz syndrome type III and their possible clinical impact. Loeys Dietz syndrome type III, caused by pathogenic SMAD3 variants, is an autosomal dominant syndrome characterized by aneurysms and arterial tortuosity in combination with osteoarthritis. Neurovascular abnormalities have been described in other heritable aortic syndromes, however, reliable data in Loeys Dietz syndrome type III is missing. In our tertiary center, all adult patients with confirmed Loeys Dietz syndrome type III are followed in a standardized aorta outpatient clinic including Computed Tomography Angiography (CTA) of the head and neck region at baseline and (tri) yearly during follow-up. We performed an analysis of the neurovascular imaging findings and clinical follow-up. The primary outcome was a combined endpoint of mortality, dissection, cerebral vascular event and intervention. In addition, tortuosity and vascular growth were assessed. In total 26 patients (mean age 38.4 years, 38.5% males) underwent 102 (mean 3.9 (1-8) per patient) neurovascular Computed Tomography Angiography scans between 2010 and 2021. In 84.6% some form of neurovascular abnormality was found. The abnormalities at baseline were aneurysm (26.9%) dissection flap (7.7%), arterial tortuosity (61.5%), arterial coiling (23.1%) and arterial kinking (3.8%). During follow up (mean 8.85 (1-11) years) one patient suffered from sudden death and one patient needed a neuro-radiological intervention. No cerebral bleeding or stroke occurred. In conclusion, neurovascular imaging in Loeys Dietz syndrome type III patients revealed abnormalities such as aneurysm, tortuosity, coiling and kinking in the vast majority of patients, but clinical events were rare. Neurovascular screening and follow up is advised in all Loeys Dietz syndrome type III patients.
Asunto(s)
Aneurisma de la Aorta/epidemiología , Arterias/anomalías , Aneurisma Intracraneal/epidemiología , Inestabilidad de la Articulación/epidemiología , Síndrome de Loeys-Dietz/patología , Fenotipo , Enfermedades Cutáneas Genéticas/epidemiología , Malformaciones Vasculares/epidemiología , Niño , Preescolar , Femenino , Humanos , Lactante , Síndrome de Loeys-Dietz/complicaciones , Síndrome de Loeys-Dietz/genética , Masculino , Proteína smad3/genéticaRESUMEN
Pompe disease is a metabolic disorder caused by a deficiency of the glycogen-hydrolyzing lysosomal enzyme acid α-glucosidase (GAA), which leads to progressive muscle wasting. This autosomal-recessive disorder is the result of disease-associated variants located in the GAA gene. In the present study, we performed extended molecular diagnostic analysis to identify novel disease-associated variants in six suspected Pompe patients from four different families for which conventional diagnostic assays were insufficient. Additional assays, such as a generic-splicing assay, minigene analysis, SNP array analysis, and targeted Sanger sequencing, allowed the identification of an exonic deletion, a promoter deletion, and a novel splicing variant located in the 5' UTR. Furthermore, we describe the diagnostic process for an infantile patient with an atypical phenotype, consisting of left ventricular hypertrophy but no signs of muscle weakness or motor problems. This led to the identification of a genetic mosaicism for a very severe GAA variant caused by a segmental uniparental isodisomy (UPD). With this study, we aim to emphasize the need for additional analyses to detect new disease-associated GAA variants and non-Mendelian genotypes in Pompe disease where conventional DNA diagnostic assays are insufficient.
RESUMEN
Analyses in our diagnostic DNA laboratory include genes involved in autosomal recessive (AR) lysosomal storage disorders such as glycogenosis type II (Pompe disease) and mucopolysaccharidosis type I (MPSI, Hurler disease). We encountered 4 cases with apparent homozygosity for a disease-causing sequence variant that could be traced to one parent only. In addition, in a young child with cardiomyopathy, in the absence of other symptoms, a diagnosis of Pompe disease was considered. Remarkably, he presented with different enzymatic and genotypic features between leukocytes and skin fibroblasts. All cases were examined with microsatellite markers and SNP genotyping arrays. We identified one case of total uniparental disomy (UPD) of chromosome 17 leading to Pompe disease and three cases of segmental uniparental isodisomy (UPiD) causing Hurler-(4p) or Pompe disease (17q). One Pompe patient with unusual combinations of features was shown to have a mosaic segmental UPiD of chromosome 17q. The chromosome 17 UPD cases amount to 11% of our diagnostic cohort of homozygous Pompe patients (plus one case of pseudoheterozygosity) where segregation analysis was possible. We conclude that inclusion of parental DNA is mandatory for reliable DNA diagnostics. Mild or unusual phenotypes of AR diseases should alert physicians to the possibility of mosaic segmental UPiD. SNP genotyping arrays are used in diagnostic workup of patients with developmental delay. Our results show that even small Regions of Homozygosity that include telomeric areas are worth reporting, regardless of the imprinting status of the chromosome, as they might indicate segmental UPiD.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Mucopolisacaridosis I/genética , Polimorfismo de Nucleótido Simple , Disomía Uniparental , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , MasculinoRESUMEN
N-acetylglutamate synthase deficiency (NAGSD, MIM #237310) is an autosomal recessive disorder of the urea cycle that results from absent or decreased production of N-acetylglutamate (NAG) due to either decreased NAGS gene expression or defective NAGS enzyme. NAG is essential for the activity of carbamylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of the urea cycle. NAGSD is the only urea cycle disorder that can be treated with a single drug, N-carbamylglutamate (NCG), which can activate CPS1 and completely restore ureagenesis in patients with NAGSD. We describe a novel sequence variant NM_153006.2:c.-3026C > T in the NAGS enhancer that was found in three patients from two families with NAGSD; two patients had hyperammonemia that resolved upon treatment with NCG, while the third patient increased dietary protein intake after initiation of NCG therapy. Two patients were homozygous for the variant while the third patient had the c.-3026C > T variant and a partial uniparental disomy that encompassed the NAGS gene on chromosome 17. The c.-3026C > T sequence variant affects a base pair that is highly conserved in vertebrates; the variant is predicted to be deleterious by several bioinformatics tools. Functional assays in cultured HepG2 cells demonstrated that the c.-3026C > T substitution could result in reduced expression of the NAGS gene. These findings underscore the importance of analyzing NAGS gene regulatory regions when looking for molecular causes of NAGSD.
Asunto(s)
N-Acetiltransferasa de Aminoácidos/genética , Elementos de Facilitación Genéticos , Variación Genética , Trastornos Innatos del Ciclo de la Urea/etiología , N-Acetiltransferasa de Aminoácidos/metabolismo , Secuencia de Bases , Niño , Preescolar , Femenino , Humanos , Hiperamonemia , Pronóstico , Trastornos Innatos del Ciclo de la Urea/metabolismo , Trastornos Innatos del Ciclo de la Urea/patologíaRESUMEN
Heterozygous gain of function mutations in the ZIC1 gene have been described with syndromic craniosynostosis, variable cerebral or cerebellar abnormalities and mild to moderate developmental delay. Deletion of chromosome 3q25.1 including both adjacent ZIC1 and ZIC4 genes have been described as a cause of variable cerebellar abnormalities including Dandy-Walker malformation. We report two siblings presenting with neonatal microcephaly, agenesis of the corpus callosum, brachycephaly with reduced volume of the posterior fossa, cerebellar and pons hypoplasia, scoliosis and tethered cord (closed neural tube defect). One of the siblings had apparent partial rhombencephalosynapsis. Trio analysis of exome sequencing data revealed a novel heterozygous frameshift mutation in ZIC1 at the end of exon 3 in one sibling and was confirmed by Sanger sequencing in both children. The mutation was not detected in DNA of both parents, which suggests parental gonadal mosaicism. We show that expression of the mutant allele leads to synthesis of a stable abnormal transcript in patient cells, without evidence for nonsense-mediated decay. Craniosynostosis was not present at birth, which explains why ZIC1 mutations were not initially considered. This severe brain malformation indicates that premature closure of sutures can be independent of the abnormal brain development in subjects with pathogenic variants in ZIC1.
Asunto(s)
Craneosinostosis/genética , Malformaciones del Desarrollo Cortical/genética , Microcefalia/genética , Factores de Transcripción/genética , Adolescente , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/fisiopatología , Cerebelo/fisiopatología , Niño , Preescolar , Craneosinostosis/fisiopatología , Femenino , Mutación del Sistema de Lectura , Humanos , Lactante , Masculino , Malformaciones del Desarrollo Cortical/fisiopatología , Microcefalia/fisiopatología , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/fisiopatología , Fenotipo , Escoliosis/genética , Escoliosis/fisiopatologíaRESUMEN
Left ventricular (LV) hypertrophy in subjects with hypertrophic cardiomyopathy (HCM) is variable, suggesting a role for modifying factors. Here, we determined whether aldosterone modulates hypertrophy in HCM. Cardiac and/or plasma aldosterone were measured in organ donors and HCM patients. The effect of the aldosterone synthase ( CYP11B2 ) C-344T polymorphism on LV mass index (LVMI) and interventricular septum thickness (IVS) was determined in 79 genetically independent subjects with HCM. Aldosterone in HCM hearts and plasma was similar to that in normal hearts and plasma. In HCM women, no associations between CYP11B2 genotype and any of the measured parameters were observed, whereas in HCM men, LVMI increased with the presence of the T allele. Similar T allele-related increases were observed for IVS. Multiple regression analysis revealed that the T allele-related effect on IVS occurred independently of renin, the ACE I/D polymorphism, the AT1-receptor A/C(1166)polymorphism and the AT2-receptor A/C(3123) polymorphism. In conclusion, circulating and cardiac aldosterone are normal in HCM, thereby arguing against selectively increased cardiac aldosterone production in HCM. Thus, the association between the CYP11B2 C-344T polymorphism and hypertrophy in HCM most likely relates to the T allele-related increases in circulating aldosterone. This finding raises the need for studies determining the benefit of aldosterone blockade in HCM.
Asunto(s)
Aldosterona/metabolismo , Cardiomiopatía Hipertrófica/metabolismo , Citocromo P-450 CYP11B2/genética , Miocardio/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cardiomiopatía Hipertrófica/genética , Citocromo P-450 CYP11B2/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
Biotinidase deficiency is a rare inherited metabolic disorder that can cause severe neurological symptoms. To prevent severe clinical presentations, it was included in the Dutch neonatal screening programme in 2007. Since then the number of cases detected has been high. This study set out to describe the incidence of the disease, the clinical and demographic characteristics of the neonates identified and the type of mutations found. In the south-western Netherlands, 304 982 neonates were screened between 2007 and 2012; and 92 were identified for further testing. Confirmatory testing revealed 6 (7%) with a profound biotinidase deficiency (<10% enzyme activity), 44 (48%) with a partial deficiency (10-30%) and 42 (46%) with normal activity (>30%). All six patients whose profound deficiency was confirmed had enzyme activities below 15% on neonatal screening. Mutation analysis was performed in 61 neonates: 5 'profound', 35 'partial' and 21 'normal'. All five 'profound' cases had two severe mutations. Comparison with the northern Netherlands showed that the frequency and types of mutation were representative for the Netherlands as a whole. The most common mutation detected was c.[1330G>C] (p.(Asp444His); 34%), which is considered to be mild, followed by three severe mutations c.[1368A>C], c.[1595C>T] and c.[1330G>C;511G>A]. Seven new mutations were identified. We conclude that neonatal screening for profound biotinidase produces a high number of false positives. Biotinidase deficiency was profound in less than 10% of cases identified. As biotinidase activity lay below 15% on neonatal screening in all such cases, the screening threshold might be reduced to 15%.
Asunto(s)
Deficiencia de Biotinidasa/genética , Pruebas Genéticas/normas , Deficiencia de Biotinidasa/diagnóstico , Reacciones Falso Positivas , Femenino , Pruebas Genéticas/estadística & datos numéricos , Humanos , Recién Nacido , Masculino , Países Bajos , Sensibilidad y EspecificidadRESUMEN
Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.
Asunto(s)
Cardiomiopatías/diagnóstico , Pruebas Genéticas/normas , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Mutación , Proteínas de Unión al Calcio/genética , Miosinas Cardíacas/genética , Cardiomiopatías/genética , Proteínas Portadoras/genética , Exoma , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Consentimiento Informado/legislación & jurisprudencia , Ensayos de Aptitud de Laboratorios/estadística & datos numéricos , Quinasas Quinasa Quinasa PAM/genética , Cadenas Pesadas de Miosina/genética , Países Bajos , Proteínas Serina-Treonina QuinasasRESUMEN
Tissue accumulation of circulating prorenin results in angiotensin generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to angiotensin-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-1 release. Here we investigated whether prorenin exerts angiotensin-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-1. PAI-1 release did occur during coincubation with angiotensinogen, suggesting that this effect is angiotensin mediated. Prorenin concentration-dependently activated p38 MAPK and simultaneously phosphorylated HSP27. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (n=4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (P<0.001 versus control), among which genes downstream of p38 MAPK and HSP27 involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-7, were overrepresented. Quantitative real-time RT-PCR of 7 selected genes (Opg, Timp1, Best5, Hsp27, pro-Anp, Col3a1, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the angiotensin II type 1 receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts angiotensin-independent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP27 pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression.
Asunto(s)
Angiotensina II/fisiología , Membranas Intracelulares/metabolismo , Miocitos Cardíacos/metabolismo , Renina/fisiología , Transducción de Señal/fisiología , Citoesqueleto de Actina/fisiología , Angiotensina II/farmacología , Animales , Células Cultivadas , Quimosina , Activación Enzimática , Precursores Enzimáticos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Ratas , Ratas Wistar , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/farmacología , Renina/metabolismo , Renina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Nearly 4000 abstracts were selected for presentation at the 77th Scientific Sessions of the American Heart Association, held in New Orleans, Louisiana, USA. The sessions were divided into basic, clinical and population science. The abstracts have been published in a supplement to Circulation (2004) 110(7).
Asunto(s)
American Heart Association , Enfermedades Cardiovasculares/terapia , American Heart Association/organización & administración , Animales , Fármacos Cardiovasculares/uso terapéutico , Enfermedades Cardiovasculares/metabolismo , Humanos , Louisiana , Trasplante de Células Madre/métodos , Estados UnidosRESUMEN
Interference with locally generated angiotensin II most likely underlies the beneficial effects of renin-angiotensin system blockers in cardiac disorders. Since renin is not synthesized in the heart, this enzyme must be sequestered from the circulation in order to allow angiotensin generation at cardiac tissue sites. This review addresses the various ways through which circulating (i.e., kidney-derived) renin may reach cardiac tissue sites, considering in particular the possibility that prorenin, the inactive precursor of renin, is involved in cardiac angiotensin generation, as the plasma concentrations of prorenin are tenfold higher than those of renin. Renin and prorenin diffuse into the cardiac interstitial space and bind to cardiac (pro)renin receptors/renin-binding proteins. One of these receptors is the mannose 6-phosphate/insulin-like growth factor II receptor. This receptor not only binds mannose 6-phosphate-containing ligands like renin and prorenin, it also internalizes these enzymes, and activates prorenin intracellularly. This process possibly represents (pro)renin clearance, since intracellular angiotensin generation could not be demonstrated following (pro)renin uptake by cardiomyocytes. Angiotensin II-mediated myocyte proliferation did occur when incubating cardiomyocytes with prorenin plus angiotensionogen. The effects of prorenin plus angiotensinogen were comparable to those of 100nmol/l angiotensin II, although the angiotensin II levels in the medium during exposure of the cells to prorenin plus angiotensinogen were <1nmol/l. This suggests that cardiac angiotensin II generation by circulating renin occurs predominantly on the cell surface. The presence of ACE and/or renin on the cell membrane, in the microenvironment of angiotensin receptors, would allow maximal efficiency of local angiotensin II generation, i.e., immediate binding of angiotensin II to its receptors with minimal loss into the extracellular space.
Asunto(s)
Angiotensinas/biosíntesis , Precursores Enzimáticos/metabolismo , Miocardio/metabolismo , Renina/metabolismo , ATPasas de Translocación de Protón Vacuolares , Animales , División Celular/efectos de los fármacos , Difusión , Humanos , Riñón/metabolismo , Mamíferos/metabolismo , Modelos Biológicos , Proteínas Musculares/metabolismo , Miocardio/citología , Especificidad de Órganos , Peptidil-Dipeptidasa A/metabolismo , Procesamiento Proteico-Postraduccional , Receptor IGF Tipo 2/metabolismo , Receptores de Superficie Celular/metabolismo , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Cardiomyocytes bind, internalize, and activate prorenin, the inactive precursor of renin, via a mannose 6-phosphate receptor (M6PR)--dependent mechanism. M6PRs couple directly to G-proteins. To investigate whether prorenin binding to cardiomyocytes elicits a response, and if so, whether this response depends on angiotensin (Ang) II, we incubated neonatal rat cardiomyocytes with 2 nmol/L prorenin and/or 150 nmol/L angiotensinogen, with or without 10 mmol/L M6P, 1 micromol/L eprosartan, or 1 micromol/L PD123319 to block M6P and AT(1) and AT(2) receptors, respectively. Protein and DNA synthesis were studied by quantifying [(3)H]-leucine and [(3)H]-thymidine incorporation. For comparison, studies with 100 nmol/L Ang II were also performed. Neither prorenin alone, nor angiotensinogen alone, affected protein or DNA synthesis. Prorenin plus angiotensinogen increased [(3)H]-leucine incorporation (+21 +/- 5%, mean +/- SEM, P<0.01), [(3)H]-thymidine incorporation (+29 +/- 6%, P<0.01), and total cellular protein (+14 +/- 3%, P<0.01), whereas Ang II increased DNA synthesis only (+34 +/- 7%, P<0.01). Eprosartan, but not PD123319 or M6P, blocked the effects of prorenin plus angiotensinogen as well as the effects of Ang II. Medium Ang II levels during prorenin and angiotensinogen incubation were <1 nmol/L. In conclusion, prorenin binding to M6PRs on cardiomyocytes per se does not result in enhanced protein or DNA synthesis. However, through Ang II generation, prorenin is capable of inducing myocyte hypertrophy and proliferation. Because this generation occurs independently of M6PRs, it most likely depends on the catalytic activity of intact prorenin in the medium (because of temporal prosegment unfolding) rather than its intracellular activation. Taken together, our results do not support the concept of Ang II generation in cardiomyocytes following intracellular prorenin activation.