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Rhabdoid tumor predisposition syndrome type-1 (RTPS1) is characterized by germline pathogenic variants in SMARCB1 and development of INI1-deficient rhabdoid tumors in early childhood. Due to its poor prognosis, the risk of subsequent tumor development and the impact of surveillance at later ages are poorly understood. We retrospectively reviewed individuals referred to the Cancer Genetics Program at The Hospital for Sick Children for SMARCB1 genetic testing and/or surveillance for RTPS1. In addition, to explore characteristics of late-onset tumors in RTPS1, a literature review was conducted. Of eighty-three individuals (55 probands and 28 family members), 12 probands and 4 family members were genetically confirmed with RTPS1. Four pediatric probands with RTPS1 underwent surveillance. An additional three individuals, including one patient with 22q11.2 distal deletion without history of tumor, one patient with negative genetic testing results but clinically diagnosed with RTPS1, and one sibling identified through cascade testing, underwent surveillance. Three patients with RTPS1 developed tumors between the ages of 9 and 17, including malignant rhabdoid tumors (N = 3), schwannomas (N = 4), and epithelioid malignant peripheral nerve sheath tumor (N = 1). Three of these lesions were asymptomatically detected by surveillance. A literature review revealed 17 individuals with RTPS1 who developed INI1-deficient tumors after age five. Individuals with RTPS1 remain at elevated risk for developing INI1-deficient tumors after the peak age of rhabdoid tumor in early childhood. Extension of surveillance beyond 5 years of age could lead to improved survival and reduced morbidity for these patients, and prospective evaluation of revised approaches will be important.
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PURPOSE: To evaluate the diagnostic utility of publicly funded clinical exome sequencing (ES) for patients with suspected rare genetic diseases. METHODS: We prospectively enrolled 297 probands who met eligibility criteria and received ES across 5 sites in Ontario, Canada, and extracted data from medical records and clinician surveys. Using the Fryback and Thornbury Efficacy Framework, we assessed diagnostic accuracy by examining laboratory interpretation of results and assessed diagnostic thinking by examining the clinical interpretation of results and whether clinical-molecular diagnoses would have been achieved via alternative hypothetical molecular tests. RESULTS: Laboratories reported 105 molecular diagnoses and 165 uncertain results in known and novel genes. Of these, clinicians interpreted 102 of 105 (97%) molecular diagnoses and 6 of 165 (4%) uncertain results as clinical-molecular diagnoses. The 108 clinical-molecular diagnoses were in 104 families (35% diagnostic yield). Each eligibility criteria resulted in diagnostic yields of 30% to 40%, and higher yields were achieved when >2 eligibility criteria were met (up to 45%). Hypothetical tests would have identified 61% of clinical-molecular diagnoses. CONCLUSION: We demonstrate robustness in eligibility criteria and high clinical validity of laboratory results from ES testing. The importance of ES was highlighted by the potential 40% of patients that would have gone undiagnosed without this test.
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Exoma , Enfermedades Raras , Humanos , Estudios Prospectivos , Secuenciación del Exoma , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Pruebas Genéticas/métodos , OntarioRESUMEN
Single neuronal imaging of a fully intact Drosophila larva is a difficult challenge for neurosciences due to the robust digging/burrowing behaviour of the Drosophila larva and the lack of intact immobilization methods at single-neuron resolution. In this paper, for the first time, a simple microfluidic device to completely immobilize the brain and the CNS of a live, fully-functioning Drosophila larva for single neuronal imaging has been demonstrated. The design of the microfluidic device contains a unique clamping feature which pins and bends the body of the larva at 1/3rd of its length from the head. This simple twist combined with the pinning mechanism not only could stop the locomotion of the larva but also could immobilize the major movement of internal organs including the CNS. The results showed that the bent trap could keep the single neuron completely inside the field of view (FOV) (50 µm × 50 µm) over 10 min of confocal imaging. The range of motion in the x- and y-axis was approximately 8 µm and 2.5 µm, respectively. This corresponds to a range of 16% and 6% along the axis of the channel and across it compared to the size of the FOV (50 µm × 50 µm). The calcium activity of the single neurons in a 3rd instar GCaMP5 larva (Cha-Gal4/CyO; UAS-GCaMP5G/TM3) was measured while its mouth region was exposed to 20 mM sodium azide (NaN3) for 5 s. The results showed that the activity of the neurons has been statistically (p < 0.0005) increased (â¼60%).
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Drosophila , Neuronas , Animales , Larva/fisiología , Neuronas/fisiología , Encéfalo/diagnóstico por imagen , Dispositivos Laboratorio en un ChipRESUMEN
We examined the utility of clinical and research processes in the reanalysis of publicly-funded clinical exome sequencing data in Ontario, Canada. In partnership with eight sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from seven laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); four were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically-relevant genes is modest, and the highest yield comes from validation of novel disease-gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses.
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Pruebas Genéticas , Humanos , Pruebas Genéticas/métodos , Ontario/epidemiología , Secuenciación del ExomaRESUMEN
PURPOSE: To facilitate robust economic analyses of clinical exome and genome sequencing, this study was taken up with the objective of establishing a framework for organizing diagnostic testing trajectories for patients with rare disease. METHODS: We collected diagnostic investigations-related data before exome sequencing from the medical records of 228 cases. Medical geneticist experts participated in a consensus building process to develop the SOLVE Framework for organizing the complex range of observed tests. Experts categorized tests as indicator or nonindicator tests on the basis of their specificity for diagnosing rare diseases. Face validity was assessed using case vignettes. RESULTS: Most cases had symptom onset at birth (42.5%) or during childhood (43.4%) and had intellectual disability (73.3%). On average, the time spent seeking a diagnosis before sequencing was 1989 days (SD = 2137) and included 16 tests (SD = 14). Agreement across experts on test categories ranged from 83% to 96%. The SOLVE Framework comprised observed tests, including 186 indicator and 39 nonindicator tests across cytogenetic/molecular, biochemical, imaging, electrical, and pathology test categories. CONCLUSION: Real-world diagnostic testing data can be ascertained and organized to reflect the complexity of the journey of the patients with rare diseases. SOLVE Framework will improve the accuracy and certainty associated with value-based assessments of genomic sequencing.
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Evaluación de Resultado en la Atención de Salud , Enfermedades Raras , Humanos , Recién Nacido , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Secuenciación del ExomaRESUMEN
Pediatric papillary thyroid carcinoma (PPTC) is clinically distinct from adult-onset disease. Although there are higher rates of metastasis and recurrence in PPTC, prognosis remains highly favorable. Molecular characterization of PPTC has been lacking. Historically, only 40% to 50% of childhood papillary thyroid carcinoma (PTC) were known to be driven by genomic variants common to adult PTC; oncogenic drivers in the remainder were unknown. This contrasts with approximately 90% of adult PTC driven by a discrete number of variants. In this study, 52 PPTCs underwent candidate gene testing, followed in a subset by whole-exome and transcriptome sequencing. Within these samples, candidate gene testing identified variants in 31 (60%) tumors, while exome and transcriptome sequencing identified oncogenic variants in 19 of 21 (90%) remaining tumors. The latter were enriched for oncogenic fusions, with 11 nonrecurrent fusion transcripts, including two previously undescribed fusions, STRN-RET and TG-PBF. Most fusions were associated with 3' receptor tyrosine kinase (RTK) moieties: RET, MET, ALK, and NTRK3. For advanced (distally metastatic) tumors, a driver variant was described in 91%. Gene expression analysis defined three clusters that demonstrated distinct expression of genes involved in thyroid differentiation and MAPK signaling. Among RET-CCDC6-driven tumors, gene expression in pediatric tumors was distinguishable from that in adults. Collectively, these results show that the genomic landscape of pediatric PTC is different from adult PTC. Moreover, they identify genomic drivers in 98% of PPTCs, predominantly oncogenic fusion transcripts involving RTKs, with a pronounced impact on gene expression. Notably, most advanced tumors were driven by a variant for which targeted systemic therapy exists. SIGNIFICANCE: This study highlights important distinctions between the genomes and transcriptomes of pediatric and adult papillary thyroid carcinoma, with implications for understanding the biology, diagnosis, and treatment of advanced disease in children.
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Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Genómica/métodos , Fusión de Oncogenes , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/patología , Transcriptoma , Adolescente , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Masculino , Pronóstico , Estudios Prospectivos , Tasa de Supervivencia , Cáncer Papilar Tiroideo/genética , Neoplasias de la Tiroides/genéticaRESUMEN
OBJECTIVE: Nemaline myopathy (NM) is a rare neuromuscular condition with clinical and genetic heterogeneity. To establish disease natural history, we performed a cross-sectional study of NM, complemented by longitudinal assessment and exploration of pilot outcome measures. METHODS: Fifty-seven individuals with NM were recruited at 2 family workshops, including 16 examined at both time points. Participants were evaluated by clinical history and physical examination. Functional outcome measures included the Motor Function Measure (MFM), pulmonary function tests (PFTs), myometry, goniometry, and bulbar assessments. RESULTS: The most common clinical classification was typical congenital (54%), whereas 42% had more severe presentations. Fifty-eight percent of individuals needed mechanical support, with 26% requiring wheelchair, tracheostomy, and feeding tube. The MFM scale was performed in 44 of 57 participants and showed reduced scores in most with little floor/ceiling effect. Of the 27 individuals completing PFTs, abnormal values were observed in 65%. Last, bulbar function was abnormal in all patients examined, as determined with a novel outcome measure. Genotypes included mutations in ACTA1 (18), NEB (20), and TPM2 (2). Seventeen individuals were genetically unresolved. Patients with pathogenic ACTA1 and NEB variants were largely similar in clinical phenotype. Patients without genetic resolution had more severe disease. CONCLUSION: We present a comprehensive cross-sectional study of NM. Our data identify significant disabilities and support a relatively stable disease course. We identify a need for further diagnostic investigation for the genetically unresolved group. MFM, PFTs, and the slurp test were identified as promising outcome measures for future clinical trials.
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Miopatías Nemalínicas/fisiopatología , Actinas/genética , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Estudios Transversales , Evaluación de la Discapacidad , Progresión de la Enfermedad , Nutrición Enteral , Femenino , Genotipo , Humanos , Lactante , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Proteínas Musculares/genética , Miopatías Nemalínicas/genética , Proyectos Piloto , Desempeño Psicomotor , Pruebas de Función Respiratoria , Sialorrea/epidemiología , Sialorrea/etiología , Traqueostomía/estadística & datos numéricos , Resultado del Tratamiento , Silla de Ruedas/estadística & datos numéricos , Adulto JovenRESUMEN
Hematopoietic cell lineages support organismal needs by responding to positional and systemic signals that balance proliferative and differentiation events. Drosophila provides an excellent genetic model to dissect these signals, where the activity of cues in the hemolymph or substrate can be traced to determination and differentiation events of well characterized hemocyte types. Plasmatocytes in third instar larvae increase in number in response to infection and in anticipation of metamorphosis. Here we characterize hemocyte clustering, proliferation and transdifferentiation on the heart or dorsal vessel. Hemocytes accumulate on the inner foldings of the heart basement membrane, where they move with heart contraction, and are in proximity to the heart ostia and pericardial nephrocytes. The numbers of hemocytes vary, but increase transiently before pupariation, and decrease by 4â¯h before pupa formation. During their accumulation at the heart, plasmatocytes can proliferate and can transdifferentiate into crystal cells. Serrate expressing cells as well as lamellocyte-like, Atilla expressing ensheathing cells are associated with some, but not all hemocyte clusters. Hemocyte aggregation is enhanced by the presence of a heart specific Collagen, Pericardin, but not the associated pericardial cells. The varied and transient number of hemocytes in the pericardial compartment suggests that this is not a hematopoietic hub, but a niche supporting differentiation and rapid dispersal in response to systemic signals.
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Colágeno Tipo IV/metabolismo , Proteínas de Drosophila/metabolismo , Hematopoyesis/fisiología , Hemocitos/fisiología , Animales , Diferenciación Celular/fisiología , Transdiferenciación Celular/fisiología , Colágeno/metabolismo , Colágeno/fisiología , Colágeno Tipo IV/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Corazón/fisiología , Hemolinfa/metabolismo , Larva/metabolismo , Metamorfosis Biológica/fisiología , Pupa/metabolismoRESUMEN
Imaging of living, intact Drosophila larvae is challenged if normal bodily function must be observed or when healthy larvae must be recovered for subsequent studies. Here, we describe a simple and short protocol that employs transient airborne chloroform or desflurane (1,2,2,2-tetrafluoroethyl difluoromethyl ether) to efficiently immobilize larvae without the use of manipulation devices, vaporizers or imaging chambers. This non-lethal method allows the use of anesthetics while allowing tracking of individual Drosophila into adulthood for follow-up experiments. At dosages sufficient to immobilize larvae, Desflurane, but not chloroform reduced the central nervous system response to auditory stimulus. Desflurane doses were sufficient to arrest the heart, however significant rapid recovery was observed. With our method, chloroform provided more rapid anesthesia but slower recovery than Desflurane. Without specialized hardware, this technique allows for repeated imaging of living Drosophila larvae.
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Anestésicos por Inhalación/administración & dosificación , Cloroformo/administración & dosificación , Desflurano/administración & dosificación , Drosophila/efectos de los fármacos , Inmovilización/métodos , Animales , Sistema Nervioso Central/efectos de los fármacos , Larva/efectos de los fármacos , Microscopía ConfocalRESUMEN
Context: Papillary thyroid carcinoma (PTC) is a common malignancy in adolescence and is molecularly and clinically distinct from adult PTC. Mutations in the DICER1 gene are associated with thyroid abnormalities, including multinodular goiter and differentiated thyroid carcinoma. Objective: In this study, we sought to characterize the prevalence of DICER1 variants in pediatric PTC, specifically in tumors without conventional PTC oncogenic alterations. Patients: Patients (N = 40) who underwent partial or total thyroidectomy and who were <18 years of age at the time of surgery were selected. Design: The 40 consecutive thyroidectomy specimens (30 malignant, 10 benign) underwent genotyping for 17 PTC-associated variants, as well as full sequencing of the exons and exon-intron boundaries of DICER1. Results: Conventional alterations were found in 12 of 30 (40%) PTCs (five BRAFV600E, three RET/PTC1, four RET/PTC3). Pathogenic DICER1 variants were identified in 3 of 30 (10%) PTCs and in 2 of 10 (20%) benign nodules, all of which lacked conventional alterations and did not recur during follow-up. DICER1 alterations thus constituted 3 of 18 (16.7%) PTCs without conventional alterations. The three DICER1-mutated carcinomas each had two somatic DICER1 alterations, whereas two follicular-nodular lesions arose in those with germline DICER1 mutations and harbored characteristic second somatic RNase IIIb "hotspot" mutations. Conclusions: DICER1 is a driver of pediatric thyroid nodules, and DICER1-mutated PTC may represent a distinct class of low-risk malignancies. Given the prevalence of variants in children, we advocate for inclusion of DICER1 sequencing and gene dosage determination in molecular analysis of pediatric thyroid specimens.
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ARN Helicasas DEAD-box/genética , Mutación , Ribonucleasa III/genética , Cáncer Papilar Tiroideo/epidemiología , Cáncer Papilar Tiroideo/genética , Adolescente , Edad de Inicio , Niño , Femenino , Dosificación de Gen , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Masculino , Nódulo Tiroideo/epidemiología , Nódulo Tiroideo/genéticaRESUMEN
Microinjection is an established and reliable method to deliver biological reagents such as transgenic constructs and drugs, to specific locations inside organisms such as the Drosophila embryo and C. elegans worm. In this paper, a simple compliant mechanism based PDMS-microinjection system has been demonstrated. Unlike conventional microinjectors, this unique system could allow one to precisely insert a long taper microneedle laterally and at various positions inside the length of the Drosophila embryo (up to 250 µm) with the resolution of 5 µm. Volumes as low as 30 pL with accuracy of ±10 pL were delivered inside the embryo via pressure pulses. The device has been used to study the effect of toxins on cardiogenesis in Drosophila embryos. Using this device, we demonstrate that the cardioblast (CB) migration velocity is modified in a dose sensitive manner to varying doses of injected sodium azide (NaN3) and, for the first time, quantify the effect of the toxin on heart assembly. Injection with 40 pL of NaN3 was shown to decrease CB migration velocity and filopodia number at concentrations above 10 mM, while embryos injected with the tracer Rhodamine B (0 mM NaN3) displayed no significant difference when compared to uninjected embryos. This device can be potentially used for other embryonic assays, which require accurate delivery of the reagents to a specific location within the embryo.