Molecular diagnosis of cystic fibrosis by RNA obtained from nasal epithelial cells.

BACKGROUND: The diagnosis of cystic fibrosis (CF) is established when characteristic clinical signs are coupled with biallelic CFTR pathogenic variants. No previously reported non-canonical splice site variants have to be considered as variants of uncertain significance unless their effect on splicing has been validated. METHODS: Two variants identified by next-generation sequencing were evaluated. We assayed their effects on splicing employing RNA analysis and real-time expression quantification from RNA obtained from the nasal epithelial cells of a patient with clinically suspected CF and of two patients with milder phenotypes (CFTR-related disorders). RESULTS: The variant c.164+2dup causes skipping of exon 2 (p.(Ser18_Glu54del)) and exon 2 plus 3 (p.(Ser18Argfs*16)) in CFTR mRNA. Exon 2 expression in the patient heterozygous for c.164+2dup was decreased to 7 % of the exon 2 expression in the controls. The synonymous variant c.1584G>A causes a partial skipping of exon 11. The exon 11 expression in the two patients heterozygous for this variant was 22 % and 42 % of that of the controls, respectively. CONCLUSION: We conclude that variant c.164+2dup affects mRNA processing and can be considered a CF-causing variant. The results of the functional assay also showed that the p.(Glu528=) variant, usually categorized as a neutral variant based on epidemiological data, partially affects mRNA processing in our patients. This finding would allow us to reclassify the variant as a CFTR-related variant with incomplete penetrance. RNA obtained from nasal epithelial cells is an easy and accurate tool for CFTR functional studies in patients with unclassified splice variants.

MiR-151a: a robust endogenous control for normalizing small extracellular vesicle cargo in human cancer.

Small extracellular vesicles (sEVs) in the blood of cancer patients contain higher amounts of tumor markers than those identified as free-circulating. miRNAs have significant biomedical relevance due to their high stability and feasible detection. However, there is no reliable endogenous control available to measure sEVs-miRNA content, impairing the acquisition of standardized consistent measurements in cancer liquid biopsy. In this study, we identified three miRNAs from a panel of nine potential normalizers that emerged from a comprehensive analysis comparing the sEV-miRNA profile of six lung and ovarian human cancer cell lines in the absence of or under different conditions. Their relevance as normalizers was tested in 26 additional human cancer cell lines from nine different tumor types undergoing chemotherapy or radiotherapy treatment. The validation cohorts were comprised of 242 prospective plasma and ascitic fluid samples from three different human tumor types. Variability and normalization properties were tested in comparison to miR-16, the most used control to normalize free-circulating miRNAs in plasma. Our results indicate that miR-151a is consistently represented in small extracellular vesicles with minimal variability compared to miR-16, providing a novel normalizer to measure small extracellular vesicle miRNA content that will benefit liquid biopsy in cancer patients.

Identification and Functional Analysis of APOB Variants in a Cohort of Hypercholesterolemic Patients.

Mutations in APOB are the second most frequent cause of familial hypercholesterolemia (FH). APOB is highly polymorphic, and many variants are benign or of uncertain significance, so functional analysis is necessary to ascertain their pathogenicity. Our aim was to identify and characterize APOB variants in patients with hypercholesterolemia. Index patients (n = 825) with clinically suspected FH were analyzed using next-generation sequencing. In total, 40% of the patients presented a variant in LDLR, APOB, PCSK9 or LDLRAP1, with 12% of the variants in APOB. These variants showed frequencies in the general population lower than 0.5% and were classified as damaging and/or probably damaging by 3 or more predictors of pathogenicity. The variants c.10030A>G;p.(Lys3344Glu) and c.11401T>A;p.(Ser3801Thr) were characterized. The p.(Lys3344Glu) variant co-segregated with high low-density lipoprotein (LDL)-cholesterol in 2 families studied. LDL isolated from apoB p.(Lys3344Glu) heterozygous patients showed reduced ability to compete with fluorescently-labelled LDL for cellular binding and uptake compared with control LDL and was markedly deficient in supporting U937 cell proliferation. LDL that was carrying apoB p.(Ser3801Thr) was not defective in competing with control LDL for cellular binding and uptake. We conclude that the apoB p.(Lys3344Glu) variant is defective in the interaction with the LDL receptor and is causative of FH, whereas the apoB p.(Ser3801Thr) variant is benign.

[Analysis of cardiovascular risk factors in the stroke code. An approach in advanced ages.] / Análisis de los factores de riesgo cardiovascular en el código ictus. Una aproximación en edades avanzadas.

OBJECTIVE: The purpose of this study was to analyze the effect of cardiovascular risk factors (CVRF) in 153 patients who had suffered a stroke in the province of Ourense and where the stroke code had been activated.Its realization is part of the purpose of the authors to influence chronic pathology to prevent this specific event. METHODS: A longitudinal, retrospective and observational study was applied to 153 patients with a mean age of 76±12 years who had presented some type of stroke. The independent variables were classified as quantitative (international normalized ratio [INR], blood pressure and glycemia) and in qualitative (atrial fibrillation [AF], consumption of anticoagulants [ACO], smoking and blood lipid levels). The dependent variables were the type of stroke, the affected artery, and patient mortality after thirty days, six months, and one year. For the qualitative variables, the non-parametric verification method of Kruskal-Wallis (K-W) and Mann-Whitney (M-W) was used for comparison of means and for Chi-square association. RESULTS: INR was associated with the type of event and mortality at six and twelve months (p<0,001). Glycemia was related in a statistically significant way both with the type of event, the affected artery and with mortality in the three periods. Both AF and OACs were associated with the type of event and the artery affected, the latter also with one-year mortality. Age was related to mortality in the three periods without becoming significant at thirty days. Being a man could be a risk factor for thirty -day mortality (OR>1) and being a woman for one-year mortality. Regarding the type of intervention performed, undergoing fibrinolysis or thrombectomy increased the risk of mortality compared to combined treatment, the relationship between thrombectomy and increased mortality being statistically significant exclusively in the six-month period. CONCLUSIONS: According to the results obtained, the prevention of cerebrovascular events and secondary mortality should focus mainly on high blood glucose levels, the consumption of anticoagulants, INR, and the presence of AF as cardiovascular risk factors. Studies with a larger sample size are needed to establish if there really is an impact on mortality based on sex, as well as to determine with greater certainty if habits such as smoking, poor diet and other factors play a relevant role.

OBJETIVO: El presente estudio tuvo como propósito analizar el efecto de los factores de riesgo cardiovascular (FRCV) en 153 pacientes de la provincia de Ourense que habían sufrido ictus, donde se había activado el código ictus. Su realización parte del propósito de los autores de incidir en la patología crónica para prevenir este evento en concreto. METODOS: Se realizó un estudio longitudinal, retrospectivo y observacional aplicado a 153 pacientes con una media de edad de 76±12 años que habían presentado algún de tipo de ictus. Las variables independientes fueron clasificadas en cuantitativas (índice internacional normalizado [INR], presión arterial y glucemia) y en cualitativas (fibrilación auricular [FA], consumo de anticoagulantes [ACO], hábito de fumar y nivel de lípidos en sangre). Las variables dependientes fueron el tipo de ictus, la arteria afectada y la mortalidad de paciente al cabo de treinta días, seis meses y un año. Para las variables cualitativas se utilizó el método de comprobación no paramétrico de Kruskal-Wallis (K-W) y Mann-Whitney (M-W) para comparación de medias y para asociación Chi-cuadrado. RESULTADOS: El INR resultó asociado con el tipo de evento así como con la mortalidad a seis y doce meses (p<0,001). La glucemia se relacionó de forma estadísticamente significativa tanto con el tipo de evento como con la arteria afectada, así como con la mortalidad en los tres periodos. Tanto la FA como los ACO se asociaron con el tipo de evento y con la arteria afecta, los últimos además con la mortalidad a un año. La edad se relacionó con la mortalidad en los tres periodos sin llegar a ser significativa a los treinta días. Ser hombre podría ser un factor de riesgo (OR>1) para la mortalidad a treinta días y ser mujer para la mortalidad a un año . En cuanto al tipo de intervención realizada someterse a fibrinolisis o a trombectomía aumentó el riesgo de mortalidad frente al tratamiento combinado, siendo estadísticamente significativa la relación entre trombectomía y aumento de mortalidad exclusivamente en el periodo de seis meses. CONCLUSIONES: Según los resultados obtenidos la prevención de eventos cerebrovasculares y mortalidad secundaria a estos se debe centrar principalmente en los niveles elevados de glucemia, el consumo de anticoagulantes, INR y presencia de FA como factores de riesgo cardiovascular. Se precisan estudios de mayor tamaño muestral para establecer si realmente existe un impacto en la mortalidad en función del sexo así como determinar con mayor certeza si hábitos como el fumar, la mala alimentación y otros factores desempeñan un papel relevante.

Broadening the phenotypic spectrum of EVEN-PLUS syndrome through identification of HSPA9 pathogenic variants in the original EVE dysplasia family and two sibs with milder facial phenotype.

EVEN-PLUS syndrome is a rare autosomal recessive disorder caused by biallelic pathogenic variants in the mitochondrial chaperone called mortalin, encoded by HSPA9. This genetic disorder, presenting with several overlapping features with CODAS syndrome, is characterized by the involvement of the Epiphyses, Vertebrae, Ears, and Nose (EVEN), PLUS associated findings. Only five individuals presenting with the EVEN-PLUS phenotype and biallelic variants in HSPA9 have been published. Here, we expand the phenotypic and molecular spectrum associated with this disorder, reporting two sibs with a milder phenotype and compound heterozygous pathogenic variants (a recurrent variant and a novel one). Also, we confirm a homozygous pathogenic variant in the family originally reported as EVE dysplasia.

Novel SLC12A2-ROS1 Fusion in Non-Small Cell Lung Cancer with a Significant Response to Crizotinib: The Importance of Choosing the Appropriate Next-Generation Sequencing Assay.

Identifying the druggable target is crucial for patients with nonsquamous advanced non-small cell lung cancer (NSCLC). This article adds to the spectrum of ROS1 fusion cases described in NSCLC. We describe a novel SLC12A2-ROS1 rearrangement that has not been previously reported in other cancers: a fusion that has clinical and radiological sensitivity to crizotinib. Fluorescence in situ hybridization detected the SLC12A2-ROS1 fusion and it was confirmed through hybrid capture-based next-generation sequencing (NGS); however, the fusion could not be detected by amplicon-based assay. The success of implementing NGS into routine clinical practice depends on the accuracy of testing. The test's methodological features should then be considered because they significantly affect the results. Given this patient's response to crizotinib, identifying patients with undescribed ROS1 fusions has important therapeutic implications. KEY POINTS: This is the first known description of an SLC12A2-ROS1 fusion. Considering the patient's clinical features and tumor response observed after crizotinib therapy, the authors confirm that this new rearrangement has relevant clinical impact for patients with non-small cell lung cancer. The success of implementing next-generation sequencing (NGS) into routine clinical practice depends on the accuracy of the testing. Different assays and NGS platforms can achieve differing results. Each assay's limitations need to be considered to ensure the quality of precision medicine in clinical practice.

Delineation of the clinical and radiological features of Stuve-Wiedemann syndrome childhood survivors, four new cases and review of the literature.

Stuve-Wiedemann syndrome (SWS; MIM 601559) is a rare autosomal recessive disease caused by mutations in the leukemia inhibitor factor receptor gene (LIFR). Common clinical and radiological findings are often observed, and high neonatal mortality occurs due to respiratory distress and hyperthermic episodes. Despite initially considered as a lethal disorder during the newborn period, in recent years, several SWS childhood survivors have been reported. We report a detailed clinical and radiological characterization of four unrelated childhood SWS molecularly confirmed patients and review 22 previously reported childhood surviving cases. We contribute to the definition of the childhood survival phenotype of SWS, emphasizing the evolving phenotype, characterized by skeletal abnormalities with typical radiological findings, distinctive dysmorphic features, and dysautonomia. Based on the typical features and clinical course, early diagnosis is possible and crucial to plan appropriate management and prevent potential complications. Genetic confirmation is advisable in order to improve genetic counseling to the patients and their families.

Familial hypercholesterolemia: A single-nucleotide variant (SNV) in mosaic at the low density lipoprotein receptor (LDLR).

BACKGROUND AND AIMS: Familial hypercholesterolemia is most frequently caused by genetic variants in the LDLR gene. Most of LDLR pathogenic variants are missense, followed by splicing and deletion/insertions variants. Mosaicism is a genetic condition in which an individual shows more than one clone of cells with different genotypes. The objective of this article was the molecular characterization of a patient with hypercholesterolemia. METHODS AND RESULTS: Genetic analysis of DNA from peripheral blood and saliva was performed by NGS, Sanger sequencing and pyrosequencing technologies. NGS analysis detected the pathogenic variant LDLR:c.1951G > T:p.(Asp651Tyr) in 9%-12% of reads. The presence of the variant was confirmed by pyrosequencing analysis. The variant found was functional characterized using an in vitro model (CHO-ldlA7 cells). Activity and expression of cell surface LDLR were measured by flow cytometry. Colocalization LDLR-Dil-LDL was detected by immunofluorescence. The LDLR activity showed 80% uptake, 50% binding and 53% expression of cell surface LDLR regarding wild type. CONCLUSIONS: Herein, we report the first case of a mosaic single nucleotide variant affecting the LDLR gene in a patient with familial hypercholesterolemia. As it has been described for other pathologies, mosaicism could be underestimated in FH and its detection will improve with the introduction of NGS technologies in the diagnostic routine.

Loss-of-function mutation of PCSK9 as a protective factor in the clinical expression of familial hypercholesterolemia: A case report.

RATIONALE: Proprotein convertase subtilisin/kexin 9 or PCSK9 is a protein whose main function is to regulate the number of low-density lipoprotein receptors (LDLR) present on the cell surface. Loss-of-function mutations in PCSK9 have been related to low LDL-cholesterol levels and a decrease in the risk of cardiovascular events. PATIENT CONCERNS: We present the case of a 27-year-old woman, offspring of a patient with familial homozygous hypercholesterolemia, who presented with mild-moderate hypercholesterolemia. DIAGNOSIS: Genetic analysis was performed by next generation sequencing using a customized panel of 198 genes. Sanger sequencing was used to confirm the presence of the variants of interest. The genetic analysis showed a pathogenic heterozygous mutation in LDLR [exon 6:c.902A>G:p(Asp301Gly)], as well as a loss-of-function heterozygous variant in PCSK9 [exon1:c.137 G>T:p.(Arg46Leu)]. The genetic analysis of the index case's mother revealed compound heterozygosity for 2 different mutations in LDLR [c.902A>G:p.(Asp301Gly); c.1646G>T:p.(Gly549Val)] in exon 6 and in exon 11, respectively, and the same loss-of-function variant in PCSK9 that had been found in her daughter [(PCSK9:exon1:c.137G>T:p.(Arg46Leu)]. The maternal grandfather of the index case presented the same genetic variants as his granddaughter. INTERVENTIONS: The index case did not receive any specific treatment for hypercholesterolemia. The loss-of-function variant in PCSK9 protected her from higher LDL-cholesterol levels, provided she kept partial activity of the LDLR. In her mother, instead, a PCSK9 inhibitor was tried but failed to achieve lipid control. The reason for this may be the complete absence in LDL receptor activity. LDL apheresis was started afterwards, resulting in adequate lipid level control. OUTCOMES: To the date, the index case has achieved to maintain adequate total and LDL-cholesterol levels without any other intervention. She has had no known cardiovascular complication. LESSONS: Loss-of-function mutations in PCSK9 could protect from developing more severe forms of hypercholesterolemia. The finding of these mutations (LDLR-PCSK9) in three consecutive generations could imply an adaptive mechanism against the development of hypercholesterolemia.

Effect of the Flipped Classroom and Gamification Methods in the Development of a Didactic Unit on Healthy Habits and Diet in Primary Education.

Currently, there are several methodological models that have broken into different disciplines of knowledge with the aim of making the teaching/learning process more dynamic, active and participatory for students. This is the case of Flipped Classroom, which is based on a mixed approach between e-learning and face-to-face teaching, as well as gamification, which bases its didactic principles on the recreational components of the games. Within this context, the aim of this research is to observe what effect the application of Flipped Classroom and gamification has in the development of motivation, autonomy and self-regulation towards learning through a didactic unit on healthy habits and diet in 202 students of 6th grade of Primary School from four different schools (public and state-subsidized) in the city of Granada (Spain). For this purpose, a methodological design was used with pre-test and post-test to check the effects of the experience on the students. The findings obtained showed that the application of these methods promoted an increase in students' motivation, as well as in their autonomy and self-regulation when facing the contents of the subject. For this reason, it is advocated that there is a need to continue promoting a quality and innovative educational practice according to the figure of the student today.

Percepciones docentes sobre el blended learning en las cooperativas de enseñanza del sur de España / Teachers' perceptions of blended learning in teaching cooperatives in southern Spain / Percepções dos professores sobre o ensino misto em cooperativas de ensino no sul da Espanha

Resumen Introducción: las nuevas metodologías emergentes y su aplicación educativa se han convertido en una realidad dentro de los espacios de aprendizaje, siendo el b-learning una de las metodologías más recurrentes durante la última década. Objetivo: el objetivo del presente trabajo radica en conocer el grado de empleo de la metodología b-learning en los docentes de las cooperativas de enseñanza. Materiales y métodos: la investigación se ha llevado a cabo a través de un estudio descriptivo y correlacional, utilizándose un cuestionario de corte ad hoc de cinco dimensiones. Resultados: los resultados muestran un incremento progresivo de la incorporación de la innovación dentro de las aulas y un ímpetu formativo por parte de los docentes en materia tecnológica. Conclusiones: los docentes perciben el blended learning cada vez de manera más positiva, a pesar de disponer de escasos recursos digitales y de encontrar dificultades en propiciar un rol activo del discente. Asimismo, se ha constatado la existencia de niveles intermedios de confianza hacia la tecnología, especialmente entre el profesorado de mayor edad.

Abstract Introduction: new emerging methodologies and their educational application have become a reality within learning spaces, with b-learning being one of the most recurrent methodologies during the last decade. Objective: the objective of this work is to know the degree of use of the b-learning methodology in the teachers of the teaching cooperatives. Materials and methods: the research was carried out through a descriptive and correlational study, using a five-dimensional ad hoc cut-off questionnaire. Results: the results show a progressive increase in the incorporation of innovation within the classrooms and a formative impetus on the part of the teachers in technological matters. Conclusions: teachers perceive blended learning more and more positively, in spite of having scarce digital resources and finding difficulties in propitiating an active role of the student. Likewise, it has been verified the existence of intermediate levels of confidence in technology, especially among older teachers.

Resumo Introdução: novas metodologias emergentes e sua aplicação educacional tornaram-se uma realidade dentro dos espaços de aprendizagem, sendo o b-learning uma das metodologias mais recorrentes na última década. Objetivo: o objetivo deste trabalho é conhecer o grau de utilização da metodologia b-learning nos professores das cooperativas de ensino. Materiais e métodos: a pesquisa foi realizada por meio de um estudo descritivo e correlacional, utilizando um questionário de corte ad hoc em cinco dimensões. Resultados. os resultados mostram um aumento progressivo na incorporação de inovação dentro das salas de aula e um impulso formativo por parte dos professores em questões tecnológicas. Conclusões: os professores percebem o blended learning de maneira cada vez mais positiva, apesar de ter recursos digitais escassos e encontrar dificuldades em propiciar um papel ativo do aluno. Da mesma forma, verificou-se a existência de níveis intermediários de confiança em tecnologia, especialmente entre os professores mais velhos.

Functional analysis of new variants at the low-density lipoprotein receptor associated with familial hypercholesterolemia.

Familial hypercholesterolemia is an autosomal dominant disease of lipid metabolism caused by defects in the genes LDLR, APOB, and PCSK9. The prevalence of heterozygous familial hypercholesterolemia (HeFH) is estimated between 1/200 and 1/250. Early detection of patients with FH allows initiation of treatment, thus reducing the risk of coronary heart disease. In this study, we performed in vitro characterization of new LDLR variants found in our patients. Genetic analysis was performed by Next Generation Sequencing using a customized panel of 198 genes in DNA samples of 516 subjects with a clinical diagnosis of probable or definitive FH. All new LDLR variants found in our patients were functionally validated in CHO-ldlA7 cells. The LDLR activity was measured by flow cytometry and LDLR expression was detected by immunofluorescence. Seven new variants at LDLR were tested: c.518 G>C;p.(Cys173Ser), c.[684 G>T;694 G>T];p.[Glu228Asp;Ala232Ser], c.926C>A;p.(Pro309His), c.1261A>G;p.(Ser421Gly), c.1594T>A;p.(Tyr532Asn), and c.2138delC;p.(Thr713Lysfs*17). We classified all variants as pathogenic except p.(Ser421Gly) and p.(Ala232Ser). The functional in vitro characterization of rare variants at the LDLR is a useful tool to classify the new variants. This approach allows us to confirm the genetic diagnosis of FH, avoiding the classification as "uncertain significant variants", and therefore, carry out cascade family screening.