RESUMEN
Newborn screening is designed for presymptomatic identification of serious conditions with effective early interventions. Clinical laboratories must perform prospective pilot studies to ensure that the analytical performance and workflow for a given screening test are appropriate. We assessed the potential to screen newborns for fragile X syndrome, a monogenic neurodevelopmental disorder, by establishing a customized, high-throughput PCR and analysis software system designed to detect fragile X mental retardation 1 gene repeat expansions from dried blood spots (DBSs). Assay precision, accuracy, sensitivity, and specificity were characterized across the categorical range of repeat expansions. The assay consistently resolved genotypes within three CGG repeats of reference values up to at least 137 repeats and within six repeats for larger expansions up to 200 repeats. Accuracy testing results were concordant with reference results. Full and premutation alleles were detected from subnanogram DNA inputs eluted from DBSs and from mixtures with down to 1% relative abundance of the respective expansion. Analysis of 963 deidentified newborn DBS samples identified 957 normal and 6 premutation specimens, consistent with previously published prevalence estimates. These studies demonstrate that the assay system can support high-throughput newborn screening programs.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/diagnóstico , Síndrome del Cromosoma X Frágil/genética , Pruebas Genéticas , Tamizaje Neonatal , Reacción en Cadena de la Polimerasa , Alelos , Femenino , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Recién Nacido , Masculino , Mosaicismo , Mutación , Tamizaje Neonatal/métodos , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Expansión de Repetición de TrinucleótidoRESUMEN
Androgens secreted by the testes bind the androgen receptor in developing target tissues to induce the expression of genes required for male sexual differentiation and development. Androgen concentration and androgen receptor levels vary in male reproductive target tissues during development. Exposure to environmental androgen antagonists during critical windows of fetal and postnatal development can inhibit male sexual development by blocking transcription of androgen-dependent genes. As the sensitivity to androgen antagonists under conditions of varying androgen concentrations and varying androgen receptor levels is unknown, we used a luciferase reporter assay to investigate the transcriptional effects of a known androgen antagonist (the vinclozolin metabolite M2) at different androgen concentrations and different androgen receptor levels. The ability of M2 to inhibit transcription was greater at lower concentrations of androgen (5alpha-dihydrotestosterone) and androgen receptor. The data were modeled to determine the dose-response surface of M2 and androgen receptor concentrations at different 5alpha-dihydrotestosterone levels and the relationship of the 3 components to the response. The model and hypothesis testing results suggest that, at 0.01 and 0.1 nM 5alpha-dihydrotestosterone concentrations within the expected in vivo range of free androgen levels during development, the response-surface shapes were similar and the interaction of the androgen receptor and M2 concentrations to the response were similarly antagonistic. Thus, two components of the developmental stage, androgen and androgen receptor concentrations, are critical for sensitivity to the inhibitory effects of an androgen antagonist.
