Evaluating the Calling Performance of a Rare Disease NGS Panel for Single Nucleotide and Copy Number Variants.

INTRODUCTION: Variant detection protocols for clinical next-generation sequencing (NGS) need application-specific optimization. Our aim was to analyze the performance of single nucleotide variant (SNV) and copy number (CNV) detection programs on an NGS panel for a rare disease. METHODS: Thirty genes were sequenced in 83 patients with hereditary spastic paraplegia. The variant calls obtained with LifeScope, GATK UnifiedGenotyper and GATK HaplotypeCaller were compared with Sanger sequencing. The calling efficiency was evaluated for 187 (56 unique) SNVs and indels. Five multiexon deletions detected by multiple ligation probe assay were assessed from the NGS panel data with ExomeDepth, panelcn.MOPS and CNVPanelizer software. RESULTS: There were 48/51 (94%) SNVs and 1/5 (20%) indels consistently detected by all the calling algorithms. Two SNVs were not detected by any of the callers because of a rare reference allele, and one SNV in a low coverage region was only detected by two algorithms. Regarding CNVs, ExomeDepth detected 5/5 multi-exon deletions, panelcn.MOPs 4/5 and only 3/5 deletions were accurately detected by CNVPanelizer. CONCLUSIONS: The calling efficiency of NGS algorithms for SNVs is influenced by variant type and coverage. NGS protocols need to account for the presence of rare variants in the reference sequence as well as for ambiguities in indel calling. CNV detection algorithms can be used to identify large deletions from NGS panel data for diagnostic applications; however, sensitivity depends on coverage, selection of the reference set and deletion size. We recommend the incorporation of several variant callers in the NGS pipeline to maximize variant detection efficiency.

Attenuated airway hyperresponsiveness and mucus secretion in HDM-exposed Igf1r-deficient mice.

BACKGROUND: Asthma is a common chronic lung disease characterized by airflow obstruction, airway hyperresponsiveness (AHR), and airway inflammation. IGFs have been reported to play a role in asthma, but little is known about how the insulin-like growth factor 1 receptor (IGF1R) affects asthma pathobiology. METHODS: Female Igf1r-deficient and control mice were intranasally challenged with house dust mite (HDM) extract or PBS five days per week for four weeks. Lung function measurements, and bronchoalveolar lavage fluid (BALF), serum, and lungs were collected on day 28 for further cellular, histological, and molecular analysis. RESULTS: Following HDM exposure, the control mice responded with a marked AHR and airway inflammation. The Igf1r-deficient mice exhibited an increased expression of the IGF system and surfactant genes, which were decreased in a similar manner for control and Igf1r-deficient mice after HDM exposure. On the other hand, the Igf1r-deficient mice exhibited no AHR, and a selective decrease in blood and BALF eosinophils, lung Il13 levels, collagen, and smooth muscle, as well as a significant depletion of goblet cell metaplasia and mucus secretion markers after HDM exposure. The Igf1r-deficient mice displayed a distinctly thinner epithelial layer than control mice, but this was not altered by HDM. CONCLUSIONS: Herein, we demonstrate by the first time that the Igf1r plays an important role in murine asthma, mediating both AHR and mucus secretion after HDM exposure. Thus, our study identifies IGF1R as a potential therapeutic target, not only for asthma but also for hypersecretory airway diseases.

Luminescent pentafluorophenyl-cycloplatinated complexes: synthesis, characterization, photophysics, cytotoxicity and cellular imaging.

Luminescent mono(pentafluorophenyl) cycloplatinated complexes [Pt(C^N-κC,N)(HC^N-κN)(C6F5)] [HC^N = Hthpy (2-(2-thienyl)pyridine) 2a, Hbt (2-phenylbenzothiazole) 2b, Hpq (2-phenylquinoline) 2c] have been prepared by C­H activation of a HC^N ligand in the corresponding [Pt(HC^N-κN)2(C6F5)2] (1a, 1b, 1c) complexes. Complexes 2 evolve in DMSO solution into solvate complexes and we present here successful routes for the synthesis of [Pt(C^N)(C6F5)(DMSO)] (C^N = thpy 3a, bt 3b). They have been fully characterized (X-ray for 1a, 1c, 2b, 3a and 3b), their electronic absorption and emission properties have been investigated and DFT and TD-DFT calculations for 1a, 1c, 2b and 3a have been carried out. Complexes 3a, 3b and [Pt(ppy)(C6F5)(DMSO)] 4 (Hppy = 2-phenylpyridine) show remarkable stability in a mixed DMSO-cellular medium and their cytotoxicity towards the human lung tumor (A549) and bronchial epithelial non-tumorigenic (NL20) cell lines has been evaluated by MTS assays. Their cellular localization in A549 and NL20 human cells and in mouse embryonic fibroblasts obtained from lungs (LMEFs) has also been investigated by fluorescence microscopy.

Efecto de una intervención sobre prescripción de citicolina por médicos de atención primaria / Effect of an intervention about prescribing citicoline on primary care physicians

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Adecuación diagnóstica en la Enfermedad Pulmonar Obstructiva Crónica / The adequacy of the diagnostic in chronic obstructive pulmonary disease

Objetivos. Valorar la adecuación a las guías clínicas (GOLD/SEPAR) del diagnóstico de los pacientes calificados de Enfermedad Pulmonar Obstructiva Crónica (EPOC). Material y métodos. Se seleccionaron todos los casos de EPOC del registro de pacientes crónicos de 28 cupos de 9 centros de salud de la provincia de Ourense, incluyéndose 382 casos donde se determinó la existencia de un diagnóstico correcto según resultados espirométricos. Se determinaron los factores asociados al diagnóstico correcto mediante regresión logística donde fueron incluidos edad, sexo, residencia (rural/urbana), tabaquismo, gravedad, nivel de seguimiento y tiempo desde el diagnóstico. Resultados. Eran varones 297 (77,7%) y 172 (45,0%) procedían del medio rural. La media (DE) de edad era 77,0 (±11,0) años, siendo al diagnóstico de 64,9 (±12,0) años y 11,5 (±8,0) años de tiempo de evolución. El 64,9% era o había sido fumador. Se diagnosticaron en atención primaria 26 casos (6,8%). El índice VEF1/CVF estaba registrado en 174 (45,5%) pacientes, siendo menor de 0,7 en 138 casos (36,1%), que se consideraron bien diagnosticados. En estos pacientes figuraba registrado el VEF1 en 125 casos (90,6%). El diagnóstico correcto se asociaba a enfermedad grave o muy grave (OR 5,2; IC95 1,5-17,4), procedencia urbana (OR 6,1; IC95 1,7-21,2) y edad igual o menor de 60 años (OR 3,7; IC95 1,3-11,2). Conclusión. En la historia clínica de atención primaria de los pacientes diagnosticados de EPOC existía escaso registro espirométrico y baja adecuación a los criterios diagnósticos aceptados en las guías de uso habitual (AU)

Objectives. To assess the adequacy to the clinical guides (GOLD/SEPAR) for the diagnosis of the patients classified as COPD. Material and methods. We selected all COPD cases in the registry of chronic patients of 28 general practitioners from 9 Health Centres in the province of Ourense (Spain). A total of 382 cases were included. Diagnostic accuracy was determined according to the results of spirometry. We identify factors associated with correct diagnosis by logistic regression which included age, gender, residence (rural/urban), smoking, severity, level of follow up and time since diagnosis. Results. Of the total number included, 297 were male (77.7%) and 172 patients (45.0%) came from rural areas. The average age was 77.0 (SD=±11.0) years, with a mean age at diagnosis of 64.9 (±12.0) years and the time from diagnosis was 11.5 (±8.0) years. Less than half (49.1%) patients had been smokers, and 13.1% still smoked. Twenty-six cases (6.8%) were diagnosed in Primary Care. The FEV1/FVC ratio was recorded in 174 (45.5%) patients, with less than 0.7 in 138 cases (36.1%), which were considered as correctly diagnosed. In these patients the FEV1 had been recorded in 125 cases (90.6%). A correct diagnosis was associated with severe or very severe disease (OR 5.2; 95% CI; 1.5-17.4), urban areas (OR 6.1; 95% CI, 1.7-21.2), and younger than 60 years (OR 3.7; 95% CI, 1.3-11.2). Conclusion. The number of spirometry results recorded in the Primary Care medical records of patients diagnosed with COPD was found to be low, and with little adaptation to the accepted diagnostic criteria in the guidelines that are used routinely (AU)

[The adequacy of the diagnostic in chronic obstructive pulmonary disease]. / Adecuación diagnóstica en la Enfermedad Pulmonar Obstructiva Crónica.

OBJECTIVES: To assess the adequacy to the clinical guides (GOLD/SEPAR) for the diagnosis of the patients classified as COPD. MATERIAL AND METHODS: We selected all COPD cases in the registry of chronic patients of 28 general practitioners from 9 Health Centres in the province of Ourense (Spain). A total of 382 cases were included. Diagnostic accuracy was determined according to the results of spirometry. We identify factors associated with correct diagnosis by logistic regression which included age, gender, residence (rural/urban), smoking, severity, level of follow up and time since diagnosis. RESULTS: Of the total number included, 297 were male (77.7%) and 172 patients (45.0%) came from rural areas. The average age was 77.0 (SD=±11.0) years, with a mean age at diagnosis of 64.9 (±12.0) years and the time from diagnosis was 11.5 (±8.0) years. Less than half (49.1%) patients had been smokers, and 13.1% still smoked. Twenty-six cases (6.8%) were diagnosed in Primary Care. The FEV(1)/FVC ratio was recorded in 174 (45.5%) patients, with less than 0.7 in 138 cases (36.1%), which were considered as correctly diagnosed. In these patients the FEV(1) had been recorded in 125 cases (90.6%). A correct diagnosis was associated with severe or very severe disease (OR 5.2; 95% CI; 1.5-17.4), urban areas (OR 6.1; 95% CI, 1.7-21.2), and younger than 60 years (OR 3.7; 95% CI, 1.3-11.2). CONCLUSION: The number of spirometry results recorded in the Primary Care medical records of patients diagnosed with COPD was found to be low, and with little adaptation to the accepted diagnostic criteria in the guidelines that are used routinely.