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1.
J Mol Diagn ; 24(7): 711-718, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35526834

RESUMEN

Copy number variants (CNVs) play important roles in the pathogenesis of several genetic syndromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experimental protocols protract diagnostic times from 3 to 15 days. Nanopore sequencing has the ability to reduce time to results for the detection of CNVs with the same resolution of current state-of-the-art diagnostic tests. Nanopore sequencing was compared to molecular karyotyping for the detection of pathogenic CNVs of seven patients with previously diagnosed causative CNVs of different sizes and cellular fractions. Larger chromosomal anomalies included trisomy 21 and mosaic tetrasomy 12p. Among smaller CNVs, two genomic imbalances of 1.3 Mb, a small deletion of 170 kb, and two mosaic deletions (1.2 Mb and 408 kb) were tested. DNA was sequenced and data generated during runs were analyzed in online mode. All pathogenic CNVs were identified with detection time inversely proportional to size and cellular fraction. Aneuploidies were called after only 30 minutes of sequencing, whereas 30 hours were needed to call small CNVs. These results demonstrate the clinical utility of our approach that allows the molecular diagnosis of genomic disorders within a 30-minute to 30-hour time frame and its easy implementation as a routinary diagnostic tool.


Asunto(s)
Trastornos de los Cromosomas , Aneuploidia , Aberraciones Cromosómicas , Trastornos de los Cromosomas/diagnóstico , Variaciones en el Número de Copia de ADN/genética , Humanos , Cariotipificación
2.
Mol Diagn Ther ; 24(5): 571-577, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32772316

RESUMEN

BACKGROUND: Formalin-fixed, paraffin-embedded brain specimens are a potentially rich resource to identify somatic variants, but their DNA is characterised by low yield and extensive degradation, and matched peripheral samples are usually unavailable for analysis. METHODS: We designed single-molecule molecular inversion probes to target 18 MTOR somatic mutational hot-spots in unmatched, histologically proven focal cortical dysplasias from formalin-fixed, paraffin-embedded tissues of 50 patients. RESULTS: We achieved adequate DNA and sequencing quality in 28 focal cortical dysplasias, mostly extracted within 2 years from fixation, showing a statistically significant effect of time from fixation as a major determinant for successful genetic analysis. We identified and validated seven encompassing hot-spot residues (found in 14% of all patients and in 25% of those sequenced and analysed). The allele fraction had a range of 2-5% and variants were absent in available neighbouring non-focal cortical dysplasia specimens. We computed an alternate allele threshold for calling true variants, based on an experiment-wise mismatch count distribution, well predicting call reliability. CONCLUSIONS: Single-molecule molecular inversion probes are experimentally simple, cost effective and scalable, accurately detecting clinically relevant somatic variants in challenging brain formalin-fixed, paraffin-embedded tissues.


Asunto(s)
Alelos , Pruebas Genéticas , Malformaciones del Desarrollo Cortical/diagnóstico , Malformaciones del Desarrollo Cortical/genética , Mutación , Serina-Treonina Quinasas TOR/genética , Análisis Mutacional de ADN/métodos , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Malformaciones del Desarrollo Cortical/cirugía , Sondas Moleculares , Reproducibilidad de los Resultados , Imagen Individual de Molécula , Serina-Treonina Quinasas TOR/metabolismo
4.
Gene ; 706: 162-171, 2019 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-31085274

RESUMEN

In clinical genetics, the need to discriminate between benign and pathogenic variants identified in patients with neurodevelopmental disorders is an absolute necessity. Copy number variants (CNVs) of small size can enable the identification of genes that are critical for neurologic development. However, assigning a definite association with a specific disorder is a difficult task. Among 328 trios analyzed over seven years of activity in a single laboratory, we identified 19 unrelated patients (5.8%) who carried a small (<500 kb) de novo CNV. Four patients had an additional independent de novo CNV. Nine had a variant that could be assigned as definitely pathogenic, whereas the remaining CNVs were considered as variants of unknown significance (VUS). We report clinical and molecular findings of patients harboring VUS. We reviewed the medical literature available for genes impacted by CNVs, obtained the probability of truncating loss-of-function intolerance, and compared overlapping CNVs reported in databases. The classification of small non-recurrent CNVs remains difficult but, among our findings, we provide support for a role of SND1 in the susceptibility of autism, describe a new case of the rare 17p13.1 microduplication syndrome, and report an X-linked duplication involving KIF4A and DLG3 as a likely cause of epilepsy.


Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Trastornos del Neurodesarrollo/genética , Adulto , Trastorno Autístico/genética , Niño , Preescolar , Endonucleasas , Epilepsia/genética , Femenino , Humanos , Lactante , Discapacidad Intelectual/genética , Cinesinas/genética , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Factores de Transcripción/genética
5.
Am J Med Genet A ; 170(12): 3258-3264, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27612164

RESUMEN

Simpson-Golabi-Behmel syndrome (SGBS) is an overgrowth syndrome and it is usually diagnosed postnatally, on the basis of phenotype. Prenatal ultrasonography may show fetal alterations, but they are not pathognomonic and most of them are frequently detectable only from the 20th week of gestation. Nevertheless, early diagnosis is important to avoid neonatal complications and make timely and informed decisions about the pregnancy. We report on four fetuses from two unrelated families, in whom the application of whole exome sequencing and array-CGH allowed the identification of GPC3 alterations causing SGBS. The careful follow up of pregnancies and more sophisticated analysis of ultrasound findings led to the identification of early prenatal alterations, which will improve the antenatal diagnosis of SGBS. © 2016 Wiley Periodicals, Inc.


Asunto(s)
Arritmias Cardíacas/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Gigantismo/diagnóstico , Cardiopatías Congénitas/diagnóstico , Discapacidad Intelectual/diagnóstico , Fenotipo , Aborto Inducido , Adulto , Arritmias Cardíacas/genética , Autopsia , Hibridación Genómica Comparativa , Exoma , Femenino , Feto , Genes Ligados a X , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Gigantismo/genética , Cardiopatías Congénitas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Discapacidad Intelectual/genética , Masculino , Mutación , Linaje , Diagnóstico Prenatal , Ultrasonografía Prenatal
6.
Am J Med Genet A ; 158A(4): 917-21, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22419483

RESUMEN

Bohring-Opitz syndrome (BOS) is a rare condition characterized by facial anomalies, multiple malformations, failure to thrive and severe intellectual disabilities. Recently, the cause was identified on the basis of de novo heterozygous mutations in the ASXL1 gene. We report on two novel cases carrying two previously undescribed mutations (c.2407_2411del5 [p.Q803TfsX17] and c.2893C>T [p.R965X]). These new data further support ASXL1 as cause of BOS and may contribute to a more precise definition of the phenotype caused by the disruption of this gene.


Asunto(s)
Anomalías Múltiples/genética , Craneosinostosis/genética , Discapacidad Intelectual/genética , Proteínas Represoras/genética , Niño , Codón sin Sentido/genética , Femenino , Mutación del Sistema de Lectura/genética , Humanos , Lactante , Masculino , Fenotipo
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