Functional Restoration of BRCA1 Nonsense Mutations by Aminoglycoside-Induced Readthrough.

BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in BRCA1 lead to inactive truncated protein products and are associated with high risk of breast and ovarian cancer. These mutations generate premature termination codons (PTCs). Different studies have shown that aminoglycosides can induce PTC suppression by promoting stop codon readthrough and restoring full-length (FL) protein expression. The use of these compounds has been studied in clinical trials for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy, with encouraging results. Here we show proof-of-concept data demonstrating that the aminoglycoside G418 can induce BRCA1 PTC readthrough and restore FL protein synthesis and function. We first demonstrate that G418 treatment restores BRCA1 FL protein synthesis in HCC1395, a human breast tumor cell line carrying the R1751X mutation. HCC1395 cells treated with G418 also recover HR DNA repair and restore cell cycle checkpoint activation. A set of naturally occurring BRCA1 nonsense variants encoding different PTCs was evaluated in a GFP C-terminal BRCA1 construct model and BRCA1 PTC readthrough levels vary depending on the stop codon context. Because PTC readthrough could generate FL protein carrying pathogenic missense mutations, variants representing the most probable acquired amino acid substitutions in consequence of readthrough were functionally assessed by a validated transcription activation assay. Overall, this is the first study that evaluates the readthrough of PTC variants with clinical relevance in the breast and ovarian cancer-predisposing gene BRCA1.

Development and evaluation of a training model for paracentetic suprapubic cystostomy and catheterization.

OBJECTIVES: Minimally invasive paracentetic suprapubic cystostomy is a technique that should be learned by all surgical trainees and residents. This study aimed to develop a self-made training model for paracentetic suprapubic cystostomy and placement of the suprapubic catheter and then to evaluate its effectiveness in training fourth-year medical students. METHODS: Medical students were divided into an experimental group receiving comprehensive training involving literature, video, and model use and a control group receiving all the same training protocols as the experimental group except without hands-on practice using the model. Each student's performance was video-recorded, followed by subjective and objective evaluations by urology experts and statistical analysis. RESULTS: All students completed the surgical procedures successfully. The experimental group's performance scores were significantly higher than those of the control group (median final performance scores of 91.0 vs. 86.8, respectively). Excellent scores were achieved by more students in the experimental group than in the control group (55% vs. 20%), and fewer poor scores were observed in the experimental group than in the control group (5% vs. 30%). CONCLUSIONS: Based on its cost-effectiveness, reusability, and training effectiveness, this paracentetic suprapubic cystostomy training model is able to achieve goals in teaching practice quickly and easily. Use of the model should be encouraged for training senior medical students and resident physicians who may be expected to perform emergent suprapubic catheter insertion at some time.

Increased Clinical Sensitivity and Specificity of Plasma Protein N-Glycan Profiling for Diagnosing Congenital Disorders of Glycosylation by Use of Flow Injection-Electrospray Ionization-Quadrupole Time-of-Flight Mass Spectrometry.

BACKGROUND: Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection-electrospray ionization-quadrupole time-of-flight mass spectrometry. METHODS: After PNGase F digestion of plasma glycoproteins, N-glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard. RESULTS: This assay differed from other N-glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary N-glycan. The majority of N-glycans in normal plasma had <1% abundance. Abnormal N-glycan profiles from 19 patients with known diagnoses of 11 different CDG subtypes were generated, some of which had previously been reported to have normal N-linked protein glycosylation by carbohydrate-deficient transferrin analysis. CONCLUSIONS: The clinical specificity and sensitivity of N-glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.

Development and evaluation of a training model for paracentetic suprapubic cystostomy and catheterization

OBJECTIVES: Minimally invasive paracentetic suprapubic cystostomy is a technique that should be learned by all surgical trainees and residents. This study aimed to develop a self-made training model for paracentetic suprapubic cystostomy and placement of the suprapubic catheter and then to evaluate its effectiveness in training fourth-year medical students. METHODS: Medical students were divided into an experimental group receiving comprehensive training involving literature, video, and model use and a control group receiving all the same training protocols as the experimental group except without hands-on practice using the model. Each student's performance was video-recorded, followed by subjective and objective evaluations by urology experts and statistical analysis. RESULTS: All students completed the surgical procedures successfully. The experimental group's performance scores were significantly higher than those of the control group (median final performance scores of 91.0 vs. 86.8, respectively). Excellent scores were achieved by more students in the experimental group than in the control group (55% vs. 20%), and fewer poor scores were observed in the experimental group than in the control group (5% vs. 30%). CONCLUSIONS: Based on its cost-effectiveness, reusability, and training effectiveness, this paracentetic suprapubic cystostomy training model is able to achieve goals in teaching practice quickly and easily. Use of the model should be encouraged for training senior medical students and resident physicians who may be expected to perform emergent suprapubic catheter insertion at some time.

Homozygous boricua TBCK mutation causes neurodegeneration and aberrant autophagy.

OBJECTIVE: Autosomal-recessive mutations in TBCK cause intellectual disability of variable severity. Although the physiological function of TBCK remains unclear, loss-of-function mutations are associated with inhibition of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Given that mTORC1 signaling is known to regulate autophagy, we hypothesized that TBCK-encephalopathy patients with a neurodegenerative course have defects in autophagic-lysosomal dysfunction. METHODS: Children (n = 8) of Puerto Rican (Boricua) descent affected with homozygous TBCK p.R126X mutations underwent extensive neurological phenotyping and neurophysiological studies. We quantified autophagosome content in TBCK-/- patient-derived fibroblasts by immunostaining and assayed autophagic markers by western assay. Free sialylated oligosaccharide profiles were assayed in patient's urine and fibroblasts. RESULTS: The neurological phenotype of children with TBCK p.R126X mutations, which we call TBCK-encephaloneuronopathy (TBCKE), include congenital hypotonia, progressive motor neuronopathy, leukoencephalopathy, and epilepsy. Systemic features include coarse facies, dyslipidemia, and osteoporosis. TBCK-/- fibroblasts in vitro exhibit increased numbers of LC3+ autophagosomes and increased autophagic flux by immunoblots. Free oligosaccharide profiles in fibroblasts and urine of TBCKE patients differ from control fibroblasts and are ameliorated by treatment with the mTORC1 activator leucine. INTERPRETATION: TBCKE is a clinically distinguishable syndrome with progressive central and peripheral nervous system dysfunction, consistently observed in patients with the p.R126X mutation. We provide evidence that inappropriate autophagy in the absence of cellular stressors may play a role in this disorder, and that mTORC1 activation may ameliorate the autophagic-lysosomal system dysfunction. Free oligosaccharide profiles could serve as a novel biomarker for this disorder as well as a tool to evaluate potential therapeutic interventions. Ann Neurol 2018;83:153-165.