Gastroesophageal pathologies are common and multifactorial in patients with type 1 diabetes (T1DM). The evaluation with endoscopy and 24 h pH esophageal monitoring is expensive and not always available in all medical centers, especially in developing countries so more cost-effective algorithms for diagnosis are required. Clinical questionnaires are easy to apply but its utility for gastroesophageal reflux disease screening in patients with long standing T1DM must be analyzed.  To evaluate the utility of the FSSG and Carlsson-Dent (CDQ) questionnaires to detect the frequency of gastroesophageal reflux disease in patients with T1DM.  Analytic cross-sectional study, included 54 randomly selected patients from the T1DM clinic in our hospital. Before their routine evaluation, were asked to answer FSSG and CDQ questionnaires, classifying them as positive with a score >8 or >4, respectively. we associated and compared the clinical and biochemical characteristics between patients with or without gastroesophageal reflux detected through questionnaires. p: <0.001). Those patients with tobacco consumption as well as those with poor glycemic control were more likely to score positive in either questionnaire.  Median age was 29 years (22-35), 67% were female (median of 16 years from diagnosis). In 39% of the patients FSSG was positive, CDQ was positive in 28%. A total of 71% of patients were taking medications to treat non-specific gastric symptoms. The concordance between questionnaires was 65% ( Patients T1DM had a high prevalence of gastroesophageal reflux disease. In those patients FSSG questionnaire detected a higher number of patients in comparison with CDQ.  Gastroesophageal Reflux Disease (GERD) occurs when the stomach contents leak backwards from the stomach into the esophagus, causing symptoms that impair the quality of life or in extreme cases may be the cause of other complications. Its prevalence varies from 10 to 40% in the adult population, but may be influenced by the population's characteristics, type of diet and the presence of other comorbidities Proper identification of GERD symptoms Patients with T1D may develop other diseases such as autoimmune gastritis, celiac or Crohn's disease whose symptoms could be confused with GERD. In order to properly diagnose those diseases, it's necessary to perform endoscopies, serum identification of autoantibodies and follow specific diagnostic algorithms by specialized gastroenterologists. These studies can be annoying to the patient, costly and most health systems limit them to severe cases or give priority to patients with other positive diagnostic tests. Therefore, it would be more practical to first rule out the diagnosis of GERD by means of an effective and easy-to-use clinical tool.et al., developed the FSSG questionnaire , arguing that CDQ detects symptoms of GERD but not exclusive of it, and that it is not useful for assessing the severity of the GERD or the treatment's response. According these authors, a FSSG score greater than 8 points has a sensitivity of 62% and specificity of 59% for the diagnosis of GERD, as corroborated by endoscopy In our country, access to the diagnostic tools and specialized medicine is very restricted due to the large volume of patients and the few available specialists, therefore gastroenterologists suggest the use of clinical scales as indicators of GERD. The clinical scales have been developed to allow proper identification of the disease. Carlsson et al., developed a questionnaire (CDQ) for this purpose and validated their findings with endoscopy and 24 h pHmetry -,Both questionnaires have been widely used and validated in different populations, including patients from our country We performed a cross-sectional analytical study in a group of patients randomly selected from the T1D Clinic of the Hospital de Especialidades del Centro Médico Nacional Siglo XXI of the Instituto Mexicano del Seguro Social (IMSS) from February to May 2015. All patients have met the T1D diagnosis criteria of the American Diabetes Association (ADA) p: <0.001.The inclusion criteria were a time since diagnosis of at least five years, visual ability to read the questionnaire questions and at academic capacity to understand the questionnaire without help. Patients with a history of gastric surgery, previous diagnosis with endoscopy, unintentional weight loss, severe or progressive dysphagia, or gastrointestinal bleeding were excluded. Clinical and biochemical data were recorded at the time of initial assessment. All patients completed the FSSG The Local Research and Ethics Committee on Health Research approved the protocol. The objectives of the study were fully explained to the participants, who gave their written informed consent. The procedures followed ethical standards according to the Declaration of Helsinki of 1975, revised in 2013.Laboratory studies were requested after an 8-hour fasting period. Glucose, cholesterol, HDL cholesterol (HDL-c) and triglycerides were determined using spectrophotometry technique 2 or Fisher's test. Receiver Operating Characteristics (ROC) curves of the different scales were performed to obtain the best cutoff point  for the detection of GERD. A p <0.05 was considered as statistically significant.Data were analyzed using the statistical package STATA(r) version 11. A Kolmogorov-Smirnov test was used to assess normality. Results are expressed as medians with interquartile ranges (RI). The results were evaluated by stratified analysis, searching for associations between the quantitative variables with a Mann-Whitney U test or Student's t test, and for qualitative variables using χFifty-four patients met the inclusion criteria and completed the questionnaire, 36 were women (67%). The median age of the studied group was 29 years (23-35 years), while the mean evolution time was 16 years (range 10-22 years). All patients had more than 5 years of diagnosis of type 1 diabetes and 72% more than 10 years of diagnosis . Their a2, 45% had normal weight, 34% were overweight and 21% were obese.Two-thirds of the patients were in intensive insulin treatment  with an average of 40 ± 15 units per day, which represents 0.74 ± 0.29 IU/kg. Only 17% of the patients had a dose greater than 1 IU/kg of weight. Four percent were on treatment with insulin pump. Despite intensive care, 14% had HbA1c <7%, 43% had HbA1c <8% (moderate control) and the rest were above this level. In addition, 36% were on lipid control goals . Additionally, one fifth of the patients had antihypertensive treatment. Mean BMI was 26.7 ± 5.1 kg/mHelicobacter pylori infection and 15% reported chronic use of antacid treatment.Only 8% of the patients had a previous diagnosis of GERD, none of the patients had a documented Thirty-nine percent of patients presented GERD-compatible symptoms using the CDQ and 28% using the FSSG questionnaire. Fourteen percent had both positive questionnaires, 8% only one positive questionnaire and 32% of the patients had both questionnaires negative. The median score using the FSSG questionnaire was 5 points (2 - 13 points) and for the CDQ the median was 1 point (0-5 points). p <0.001). Patients with GERD according to CDQ had higher HbA1c levels, compared to patients with negative questionnaires . In addition, smokers were more likely to be positive (p= 0.035).Considering the cutoff points reported in the literature (more than 8 points for FSSG and more than 4 points for CDQ), 39% had GERD symptoms using the FSSG and 29% using the CDQ. The kappa concordance test between the two questionnaires was 65%  of 0.514. A score of 8 points (as described in the literature) had a sensitivity of 71%, but the specificity decreased to 19%. A CDQ score of 7 points had a sensitivity of 83%, a specificity of 13% and an AUC of 0.441; meanwhile a score of 4 points (as described in the literature) had a sensitivity of 83%, with specificity of 3%.p= 0.030). Other clinical or biochemical parameters were not different among patients with or without GERD symptoms, as shown in On the other hand, patients with neuropathy were more frequently classified as positive for GERD using the FSSG . Clinical scales for GERD have been widely used and validated in different populations et al. p= 0.006). However, as specified by the authors, none of the manuscripts used for this analysis distinguish between patients with T1D or T2D. Patients with T1D are rarely studied as a separate population and this is especially important in adult patients that had diabetes for a long time and may have more severe comorbidities. Our investigation group considers that distinguishing these patients from other types of diabetes is important because, as we see in the previous results, most adult patients with T1D are very young (around 30 years of age) and they present with multiple complications and even incapacities, when they should be living normal lives with a better quality of life. Gastroesphageal diseases may be one of the contributing factors for poor metabolic control and quality of life, however these asseverations require further studies. Regarding this point, a recently published meta-analysis by Sun Although the phenotype of hyperglycemia is similar in patients with T1D and T2D, patients living with T1D have a higher frequency of autoimmune diseases et al.  Another important issue is that nowadays there are several available questionnaires that evaluate the presence of gastrointestinal symptoms. The use of each questionnaire may have pros and cons. Selecting one questionnaire over the other requires careful examination of the characteristics of the evaluated group and the questionnaire's properties and previous validation in similar populations. In this study, we found that both questionnaires detected GERD with similar frequency, with a concordance of 65%. This concordance is greater than that reported among other questionnaires. Contreras-Omaña Despite those differences, no study has demonstrated that a questionnaire is superior to another to assess the symptoms of GERD, since they were designed for different purposes in different populations. However, the use of multiple scales increases the accuracy of the results. The CDQ was created by first-contact physicians and has the advantage of being easy to apply and has been validated in different populations; however it has a relatively complex scoring system and the limitation of not being able to be used if the patient is under treatment ,et al.The FSSG questionnaire has been used in different ethnic groups, correlating adequately with endoscopic findings and has the advantage of being able to be used in patients undergoing treatment In this study, we found that a large proportion of patients with T1D had GERD symptoms detected by both questionnaires. Despite this many had not been previously evaluated by a gastroenterologist and had received empiric and irregular treatment with proton pump inhibitors or H2 blockers. It is interesting to note that patients classified as positive (with one or both questionnaires) were not different from patients with negative results with respect to time of diagnosis or glycemic control. In addition, other clinical, physical or biochemical characteristics were not different. Those findings could imply that although diabetes and its comorbidities increase the chances of having symptoms of GERD, they are not the only factors that influence its presence or severity. It could also mean that clinical assessment is not sufficient to determine which patients are at increased risk for GERD and that special questionnaire and routine evaluations by specialists are needed, particularly in those patients who are symptomatic. Finally, we found that the cutoff points proposed by the original authors lack specificity in these patients, and in order to achieve similar sensitivities and specificities, patients with T1D may need to have higher scores to suspect GERD, this may be due to other comorbidities or the fact that many patients were being empirically treated with antacid therapies. et al. H. pylori infection as well as increased prevalence of GERD symptoms in patients with T1D in comparison with healthy controls. We should remember that our population has previously reported high prevalences of H. pylori infection. They also found a correlation with HbA1c and other parameters of glycemic control and anthropometry. Similar to our study, they found a lack of association between the time since diagnosis, the presence of diabetic comorbidities and GERD symptoms. Finally, they concluded that patients with T1D had an increase in the prevalence of GERD due to a possible relationship with other related pathologies.Faria The limitations of our study include the small number of patients whose diagnosis was corroborated by endoscopy and the absence of esophageal pHmetry determinations. However, our goal was to evaluate the utility of two clinical scales to detect symptoms of GERD in order to avoid invasive procedures such as endoscopy. We believe that in developing countries, where access to specialized medical care is limited as well as funding for research, the use of cheap and simple tools such as these questionnaires is especially important in order to detect those patients that require additional care. The combination of at least two questionnaires may increase the positive predictive value, but this needs further evaluation. We probed that CDQ and FSSG are useful for detecting GERD symptoms in patients with T1D and considering the high frequency of symptomatic patients, we suggest that additional research is needed in this group of patients to determine the most effective and efficient techniques for diagnosis in order to prevent future gastrointestinal complications and reduce related costs.CDQ and FSSG questionnaires are useful for identifying GERD symptoms in patients with T1D. In this population, the FSSG questionnaire had greater power of detection for GERD in comparison with CDQ. We suggest that patients with T1D who are positive for one or both questionnaires require corroboration by endoscopic study and assessment by a gastroenterologist. La Enfermedad por Reflujo Gastroesofágico (ERGE) se produce cuando el reflujo del contenido del estómago ocasiona síntomas que deterioran la calidad de vida de los pacientes o provocan complicaciones. Tiene una prevalencia del 10 al 40% en la población adulta, pero varía de acuerdo a las características de la población, la dieta y la presencia de comorbilidades La identificación adecuada de los síntomas de ERGE Por otra parte, los pacientes con DM1 pueden desarrollar otras enfermedades como la gastritis autoinmune, la enfermedad celíaca o la enfermedad de Crohn cuyos síntomas se confunden con los de ERGE. El estudio diferencial de estas patologías incluye endoscopias, identificación sérica de autoanticuerpos y el cumplimiento de algoritmos específicos de diagnóstico realizados por gastroenterólogos especializados. Estos estudios pueden ser molestos para el paciente, costosos y en algunos países existen limitaciones para su solicitud. Por ello sería más práctico descartar en primer lugar el diagnóstico de ERGE mediante una herramienta clínica eficaz y de fácil aplicación. Ya que el estudio de ERGE no está disponible en todas las regiones de nuestro país, los gastroenterólogos sugieren que el uso de datos clínicos pueden ser buenos indicadores de ERGE. Con el paso del tiempo, se han elaborado escalas clínicas para ayudar a la adecuada identificación de la enfermedad. Carlsson et al., desarrollaron un cuestionario (CDQ) y validaron sus hallazgos con endoscopia y pHmetría de 24 horas -,Ambos cuestionarios han sido ampliamente usados y validados en diferentes poblaciones, incluyendo pacientes de nuestro país Se realizó un estudio transversal, analítico, en un grupo de pacientes seleccionados al azar de la Clínica de DM1 del Hospital de Especialidades de Centro Médico Nacional Siglo XXI del Instituto Mexicano del Seguro Social (IMSS) de febrero a mayo del 2015. Todos los pacientes cumplieron los criterios de la American Diabetes Association (ADA) para el diagnóstico de DM1 p: < 0.001.Para su inclusión debían de tener un tiempo de diagnóstico de al menos cinco años, capacidad visual para leer las preguntas del cuestionario y al menos escolaridad primaria para comprender el cuestionario sin ayuda. Se excluyeron a los pacientes con antecedente de cirugía gástrica, diagnóstico previo por endoscopia, pérdida de peso no intencionada, disfagia severa o progresiva o hemorragia gastrointestinal. Se registraron los datos clínicos y bioquímicos al momento de la valoración inicial. Todos los pacientes completaron los cuestionarios FSSG El protocolo fue aprobado por el Comité Local de Investigación y Ética en Investigación en Salud. Los objetivos del estudio fueron ampliamente explicados a los participantes, quienes dieron su consentimiento informado por escrito. Los procedimientos respetaron las normas éticas de acuerdo a la Declaración de Helsinki de 1975, revisada en 2013. Se solicitaron estudios de laboratorio a los participantes después de un periodo de 8 horas de ayuno. Se determinó glucosa, colesterol, colesterol HDL (c-HDL) y triglicéridos, mediante técnica de espectrofotometría 2 o prueba de Fisher. Se realizaron curvas ROC (Receiver Operating Characteristics) de las diferentes escalas para obtener el mejor punto de corte  para la detección de ERGE.. Se consideró p < 0.05 como estadísticamente significativo. Los datos fueron analizados utilizando el paquete estadístico STATA (r) versión 11. Se utilizó la prueba de Kolmogorov-Smirnov para valorar normalidad. Los resultados son expresados como medias y desviaciones estándar (DE) o medianas con rangos intercuartílicos (RI), de acuerdo a la distribución de los datos. Los resultados fueron evaluados mediante un análisis estratificado, buscando asociaciones entre las variables cuantitativas mediante la prueba de U de Mann-Whitney o t de Student y para variables cualitativas mediante χCincuenta y cuatro pacientes cumplieron con los criterios de inclusión y completaron el cuestionario, 36 mujeres (67%). La mediana de edad del grupo estudiado fue de 29 años (23-35 años), mientras que el tiempo medio de evolución fue de 16 años (rangos 10-22 años). Todos los pacientes tuvieron más de 5 años de diagnóstico de diabetes tipo 1 y 72% más de 10 años de diagnóstico . Las comDos tercios de los pacientes utilizaron un régimen intensivo  con un promedio de 40 ± 15 unidades por día, que representa una media de 0.74 ± 0.29 UI/kg. Solo 17% de los pacientes tuvieron una dosis mayor a 1 UI/kg de peso. El 4% estaban en tratamiento con bomba de insulina. A pesar de la terapia intensiva, 14% presentaron HbA1c <7%, 43% estaban con HbA1c <8% (control moderado) y el resto estaban por arriba de este nivel. Además, 36% estaban en metas para el control de lípidos en pacientes diabéticos  . Adicionalmente, un quinto de los pacientes tuvieron tratamiento antihipertensivo. 2, 45% de los pacientes presentaron peso normal, 34% sobrepeso y 21% obesidad.El IMC reportado fue de 26.7 ± 5.1 kg/mHelicobacter pylori y 15% reportaron uso crónico de tratamiento antiácido. Solo el 8% de los pacientes tuvieron diagnóstico previo de ERGE, ningún paciente presentó infección por Los pacientes que presentaron síntomas compatibles con ERGE utilizando el CDQ fue de 39% y 28% utilizando el cuestionario FSSG;.para ambos cuestionarios positivos se presentaron el 14% de los pacientes, 8% sólo un cuestionario positivo y 32% de los pacientes tenían ambos cuestionarios negativos. La mediana de puntaje utilizando el cuestionario FSSG fue de 5 puntos (2-13 puntos) y para el CDQ la mediana fue de 1 punto (0-5 puntos).p <0.001). Los pacientes con ERGE de acuerdo al CDQ tenían medianas de concentraciones de HbA1c más altas, en comparación con los pacientes con cuestionarios negativos . Además, los fumadores eran más propensos a resultar positivos (p= 0.035).Considerando los puntos de corte reportados en la literatura (más de 8 puntos para FSSG y más de 4 puntos para CDQ), 39% tenían síntomas de ERGE utilizando el FSSG y 29% utilizando el CDQ. El test de concordancia kappa entre ambos cuestionarios fue del 65%  de 0.514. Mientras que un puntaje de 8, la sensibilidad fue del 71%, pero la especificidad disminuyó al 19%. Con el CDQ, a 7 puntos se tiene una sensibilidad del 83%, una especificidad del 13% y una AUC de 0.441; mientras que con 4 puntos (descritos en la literatura) se alcanzó una sensibilidad del 83%, con especificidad del 3%.p= 0.030). También se observó que los pacientes fumadores tenían mayor frecuencia de sintomatología (p= 0.04). Otros parámetros clínicos o bioquímicos no fueron diferentes entre los pacientes con o sin síntomas de ERGE, como se observa en la Por otra parte, los pacientes con neuropatía fueron más frecuentemente clasificados como positivos para ERGE utilizando el FSSG . Las escalas clínicas para ERGE han sido ampliamente usadas y validadas en diferentes poblaciones et al. p= 0.006); sin embargo, tal como lo especifican los autores, ninguna de las publicaciones utilizadas para este análisis hace la distinción entre pacientes con DM1 o DM2. Los pacientes con DM1 raramente son estudiados como una población por separado, especialmente tratándose de pacientes adultos, con largo tiempo de evolución y con comorbilidades más severas. Si bien el fenotipo de hiperglucemia es similar en los pacientes con DM1 y DM2, los pacientes que viven con DM1 tienen mayor frecuencia de enfermedades autoinmunes et al. Con respecto a esto, un meta-análisis recientemente publicado por Sun et al. A pesar de estas diferencias, no existen estudios que demuestren que un cuestionario es superior a otro para valorar los síntomas de ERGE, ya que fueron diseñados para propósitos diferentes en poblaciones diferentes; sin embargo, el uso de múltiples escalas incrementa la exactitud de los resultados. El CDQ fue creado para y por médicos de primer contacto, tiene la ventaja de ser fácil de aplicar y ha sido validado en diferentes poblaciones; sin embargo, tiene un sistema de puntaje relativamente complejo y la limitante de no poder ser utilizado si el paciente está en tratamiento ,et al.El cuestionario FSSG ha sido utilizado en diferentes grupos étnicos con buenos resultados, con buena correlación entre el puntaje y los hallazgos endoscópicos y tiene la ventaja de poder ser utilizado en los pacientes en tratamiento En este estudio, encontramos que una gran proporción de pacientes con DM1 tenían síntomas de ERGE detectados mediante ambos cuestionarios; a pesar de esto, muchos no habían sido previamente evaluados por un gastroenterólogo y habían recibido tratamiento empírico e irregular con inhibidores de la bomba de protones o bloqueadores H2. Es interesante mencionar que los pacientes clasificados como positivos (con uno o los dos cuestionarios) no fueron diferentes de los pacientes con resultados negativos, con respecto al tiempo de diagnóstico o el control glucémico. Además, otras características clínicas, físicas o bioquímicas no fueron diferentes; lo cual podría traducirse en que si bien la diabetes y sus comorbilidades incrementan las posibilidades de tener síntomas de ERGE, no son el único factor que influye en su presencia o severidad. También puede significar que la valoración clínica no es suficiente para determinar que pacientes tienen mayor riesgo de presentar ERGE y que se necesitan cuestionarios especiales y evaluaciones de rutina por especialistas, sobre todo en aquellos pacientes sintomáticos. et al. H. Pylori, así como incremento en la prevalencia de síntomas de ERGE en pacientes con DM1 en comparación con controles sanos y una posible correlación con HbA1c y otros parámetros de control glucémico y antropometría. En concordancia con nuestro estudio, también encontraron falta de asociación entre el tiempo de diagnóstico, la presencia de comorbilidades diabéticas y los síntomas de ERGE. Finalmente, concluyeron que los pacientes con DM1 tenían incremento en la prevalencia de ERGE por una posible relación con otras patologías asociadas a DM1. Faria Entre las limitaciones de este estudio, radica el escaso número de pacientes en quienes se corroboró el diagnóstico mediante endoscopia y la ausencia de determinaciones de pHmetría esofágica; sin embargo, nuestro objetivo era evaluar la utilidad de dos escalas clínicas para detectar síntomas de ERGE con el fin de evitar procedimientos invasivos como la endoscopia. Nosotros consideramos que en países en vías de desarrollo, donde el acceso a atención médica especializada es limitado y la obtención de fondos para investigación es difícil, el uso de herramientas económicas y sencillas como estos cuestionarios, son especialmente útiles para detectar qué pacientes requieren atención adicional o inmediata. La combinación de al menos dos cuestionarios puede incrementar el valor predictivo positivo, lo cual aún necesita ser evaluado. Consideramos que el CDQ y el FSSG son útiles para detectar síntomas de ERGE en pacientes con DM1 y, considerando la elevada frecuencia de pacientes sintomáticos, sugerimos que se necesita investigación adicional en este grupo de pacientes para determinar cuáles son las técnicas más efectivas y eficaces para el diagnóstico, con la finalidad de prevenir futuras complicaciones gastrointestinales y disminuir los costos relacionados.Los cuestionarios CDQ y FSSG son útiles para identificar síntomas de ERGE en pacientes con DM1. En esta población, el cuestionario FSSG tuvo mayor poder de detección de ERGE en comparación con el CDQ. Sugerimos que los pacientes con DM1 que resulten positivos para uno o ambos cuestionarios, requieren corroboración mediante estudio endoscópico y valoración por un gastroenterólogo.


Affiliation 1 is incomplete. It should read: 1. Department of Experimental Medicine, School of Medicine, Universidad Nacional Autónoma de México (UNAM), Hospital General de México, Mexico City, Mexico.


The first sentence in the Acknowledgments section should read: "We are indebted to Mohamed Sakho, Barré Soropogui, Amadou Doré and Fodé Kourouma for their contribution in the fieldwork."


We regret any confusion or inconvenience this error may have caused.In the article "Prenatal HIV Testing in the US-Mexico Border Region, 2005: The Brownsville-Matamoros Sister City Project for Women's Health," author information for Ginger L. Gossman was omitted. At the time of the study, Dr Gossman was affiliated with the Family Health Research and Program Development, Office of Title V and Family Health, Texas Department of State Health Services, Austin, Texas. The correction was made to our Web site on September 15, 2008, and appears online at http://www.cdc.gov/pcd/issues/2008/oct/08_0106.htm. Lamentamos cualquier inconveniente o confusión que este error haya podido causar.En el artículo "Realización de la Prueba Prenatal de VIH en la Región Fronteriza México-Estados Unidos, 2005: El Proyecto Para la Salud de la Mujer de las Ciudades Hermanas de Matamoros-Brownsville," se omitió la información de la autora Ginger L. Gossman. Al momento del estudio, la Dra. Gossman estaba afiliada a la oficina de Investigación de Salud Familiar y Desarrollo de Programas, Oficina de Título V y Salud Familiar, Departamento Estatal de Servicios de Salud de Texas, Austin, Texas. La corrección se hizo en nuestro sitio Web el 15 de septiembre de 2008 y aparece en la siguiente dirección de Internet:


Dr. Hugues Tolou should be included in the byline as the eighth author. He is affiliated with the Unité de Virologie Tropicale, Institut de Médecine Tropicale du Service de Santé des Armées, Marseille, France. His contributions were as follows: Contributed reagents/materials/analysis tools.


It is relevant to elucidate the influence that mean linear endovenous energy density (LEED) has on the success of endovenous laser ablation treatment for chronic venous insufficiency, in order to reduce the method’s adverse effects.To evaluate the influence of mean LEED on the prevalence of saphenous closure 30 days after the laser ablation procedure.153 lower limbs from 118 patients seen at a tertiary hospital and treated for chronic venous insufficiency with endovenous 1470 nm laser ablation under local anesthesia were evaluated. The mean LEED used to treat patients was calculated to determine whether greater than average LEED was required for treatment success.A significant difference (p = 0.021) in saphenofemoral junction closure was associated with mean LEED used above the knee. Conversely, there was no significant difference in the thigh segment.Linear intravenous energy density greater than the mean of 70.57 J/cm was associated with a higher rate of closure at the saphenofemoral junction. However, density did not have an influence on the result for the thigh segment, showing that an energy density exceeding 70.57 J/cm tends not to be required for treatment of this segment. Chronic venous insufficiency (CVI) is a common disease in the vascular surgeon’s office, affecting 25 to 33% of women and 10 to 20% of men in the adult Western population.-Treatments for CVI include: clinical management and lifestyle changes; foam sclerotherapy and varicose vein surgery using the mini-incisions technique; saphenectomy by fleboextraction; and endoluminal methods such as radio frequency and laser thermoablation .,,,The minimally invasive techniques are alternatives to conventional surgical treatment with great saphenous vein (GSV) stripping and they have been increasingly adopted within medical practice.In view of the growing acceptance of EVLA as an alternative method to stripping for treatment of CVI,This was a retrospective study, that reviewed medical charts for 193 patients with CVI seen at a tertiary hospital, treated with EVLA, and reassessed 30 days later. The project was approved by the institutional Ethics Committee and registered under protocol number 80030817.0.00005580 on the Plataforma Brasil.The inclusion criteria were patients with CVI treated with EVLA from the saphenofemoral junction (SFJ) to the knee at the Hospital Angelina Caron from 2011 to February of 2018. Exclusion criteria were: patients who did not return for the 30-day follow-up; patients with peripheral arterial disease, severe hypercoagulation syndromes, or a history of deep venous thrombosis or deep venous insufficiency; and patients with incomplete medical records.®16, preferably at a point distal of the last tributary vein with reflux, followed by insertion of a 6F valved introducer. Next, the optical fiber  was inserted and connected to an endovenous laser energy source with a standard wavelength of 1470 nm .A total of 153 lower limbs were analyzed from 118 patients who had been treated with EVLA for CVI. The procedure was initiated with the patient in decubitus dorsal. Anesthetic was applied to the puncture site and ultrasound-guided puncture of the GSV was conducted using an AbocathOnce the endoluminal position within the GSV at 2.5 cm distal of the SFJ had been confirmed, tumescent local anesthesia was administered along the course of the GSV being treated. A syringe or mechanical infusion pump was used to administer a solution comprising 250 mL 0.9% saline to 20 mL of 2% lidocaine and adrenaline 1:100,000 with 4 mL of 8.4% sodium bicarbonate . Once anAfter the procedure, dressing was applied, exerting extrinsic compression along the path of the GSV, using cotton wool pads and 7/8 high compression elastic stockings (30-40 mmHg). Patients were discharged from hospital on the same day as the operation, after approximately 2 hours of postoperative observation, with a 5-day prescription for analgesics and nonsteroidal anti-inflammatories. The elastic stockings were left in place for 48 hours and removed by the patients themselves. From the third day onwards, the patient wore the stockings during the day and removed them for bathing and sleeping. Although there were no specific restrictions on walking, patients were advised to spend around 40 minutes of each hour at rest with legs raised.Patients were reassessed at 7 and 30 days after surgery, with physical examination and Doppler vascular echography . The mosUltrasound assessment of absence of reflux at the SFJ followed the recommendations contained in the clinical practice guidelines for patients with varicose veins and venous diseases published by the Society for Vascular Surgery and the American Venous Forum. A positive result was defined as absence of patent GSV stump and no reflux. For analysis, patients were classified as absence of patent stump, partially patent stump, or presence of reflux .Data were collected from medical records and stored in a Microsoft Excel spreadsheet. Data analysis was conducted with the help of SPSS, version 22.0.t test for parametric variables and the Mann-Whitney test for nonparametric variables. Results with p values below 0.05 were considered significant.Results were expressed using descriptive statistics. Inferential analysis used Pearson’s chi-square test, Fisher’s exact test, Student’s A total of 193 patient records were analyzed and 118 records and 153 lower limbs were selected for analysis. The majority of the study population were female (55.9%) and overweight  with relation to mean LEED administered, according to analysis of variance (ANOVA), with p = 0.021 . The BonWith relation to the type of fiber used and the LEED administered, there was a trend towards significant difference, according to the Levine test, with p = 0.084. According to this test, the linear fiber needed a higher LEED, of 73.30, compared to mean LEED of 69.21 for the radial fiber. When saphenous closure and presence of reflux were compared by type of fiber used, there was no significant difference (p = 0.174), according to the chi-square test.-Chronic venous insufficiency can be defined as a condition of the superficial and/or deep venous system. The disease’s prevalence has been growing worldwide and increases with age. A study conducted with a population from Botucatu, in the Brazilian state of São Paulo, reported a 35.5% prevalence of varicose veins.Currently, studies investigating EVLA are attempting to identify safer ways of employing it, with fewer adverse effects. They analyze variables such as the wavelength and energy employed, the velocity at which the laser is applied, and different types of optical fibers.,The laser light emitted from the generator causes a thermal reaction in which the laser energy delivered is captured by the intracellular water in the endothelial cells of the saphenous vein. This mechanism of action can be regulated by physical parameters, such as wavelength, method of energy administration, and quantity of energy per surface area.-,-The level of energy absorbed is extremely important, since the vessel will only retract once it reaches a certain level and, if this is not achieved, the vein may remain open or recanalize.The term LEED was first used in 2005 and became the reference for calculation of the energy used in the procedure because of its simplicity and the possibility of standardization. The formula used takes into account the power used and the velocity at with the optical fiber is tractioned.In the lower limbs assessed in this study, mean LEED was 70.57 J/cm and it was observed that LEED exceeding this mean was necessary to achieve effective thermal ablation at the SFJ. The higher energy level provoked partial or complete obliteration at the SFJ and an absence of reflux. Similar results were observed by Proebstle et al.At 30 days follow-up of patients treated for CVI under local anesthesia with EVLA, it was observed that at the SFJ using a LEED exceeding a mean of 70.57 J/cm was associated with better results, with higher rates of obliteration and absence of reflux, in relation to lower LEED. There was no significant difference in the thigh segment. This prompts the conclusion that for this segment it is not necessary to employ such a high energy level in order to achieve the desired results, and it is therefore possible to employ a lower LEED to reduce the chance of complications.Although these results are compatible with the literature, further studies are needed with data specifically relating to the LEED applied at the SFJ and in the thigh, and their relationship with the incidence of paresthesia. A insuficiência venosa crônica (IVC) é uma doença comum no consultório do cirurgião vascular e ocorre em 25 a 33% das mulheres e em 10 a 20% dos homens na população adulta ocidentallaser -O tratamento da IVC compreende: medidas clínicas e mudança de hábitos de vida; escleroterapia com espuma e cirurgia de varizes através da técnica de mini-incisões; safenectomia por fleboextração; e métodos endoluminais como radiofrequência e termoablação a stripping da veia safena magna (VSM) e vêm ganhando espaço na medicina,,,As técnicas minimamente invasivas são alternativas ao tratamento convencional cirúrgico de stripping para o tratamento da IVClinear endovenous energy density, LEED) no desfecho do procedimento após 30 dias.Diante da crescente valorização da EVLA como um método alternativo ao Trata-se de um estudo retrospectivo que avaliou prontuários de 193 pacientes com IVC de um hospital referência, submetidos a EVLA e reavaliados após 30 dias. O projeto foi aprovado pelo Comitê de Ética da nossa instituição, sob o registro nº 80030817.0.00005580 na Plataforma Brasil.Os critérios de inclusão foram pacientes com IVC submetidos a EVLA desde a junção safenofemoral (JSF) até o joelho no HAC de 2011 até fevereiro de 2018. Já os critérios de exclusão foram: pacientes que não retornaram em 30 dias; pacientes com doença arterial periférica, síndromes de hipercoagulação grave e história de trombose venosa profunda ou de insuficiência venosa profunda; e pacientes com prontuários incompletos.®16, preferencialmente em um ponto distal à última veia tributária com refluxo, seguida de instalação de introdutor valvulado 6F. A seguir, a fibra ótica  era introduzida e conectada a um gerador de energia a laser endovenoso com comprimento de onda padrão de 1470 nm e registrado na Agência Nacional da Vigilância Sanitária (80058580018).Foram então analisados 153 membros inferiores de 118 pacientes submetidos ao tratamento com EVLA para IVC. O procedimento iniciava-se com o paciente em decúbito dorsal. Era realizado um botão anestésico no local da punção e procedia-se com punção ecoguiada da VSM com Abocathlaser . A alta hospitalar se dava no mesmo dia, depois de aproximadamente 2 horas de observação após o término da cirurgia, com prescrição de analgésicos e anti-inflamatórios não esteroidais por 5 dias. A meia elástica era mantida por 48 horas e retirada pelo próprio paciente. A partir do terceiro dia, o paciente vestia as meias durante o dia e as retirava durante o banho e para dormir. Embora não houvesse restrição à deambulação, recomendava-se cerca de 40 minutos de repouso com pernas elevadas a cada hora.Os pacientes foram reavaliados aos 7 e 30 dias após a cirurgia através de exame físico e ecografia vascular com Doppler . Entre oSociety for Vascular Surgery e da American Venous Forum. Considerou-se resultado positivo a ausência de coto pérvio e de refluxo. Além disso, os pacientes foram divididos em ausência de coto pérvio, coto parcialmente pérvio e presença de refluxo  e apresentava sobrepeso , e a idade média foi de 55,35 anos. Foi utilizada fibra radial dois anéis em 66,8% e linear em 33,2% dos pacientes, todas com 600 µ de diâmetro e potência de 6 watts. Os dados descritivos são apresentados abaixo em forma de tabelas referentes à idade e ao IMC , ao sexoHouve diferença significativa entre os grupos  com relação à LEED média aplicada, segundo a análise de variância (ANOVA), com p = 0,021 . O pós-tEm relação ao tipo de fibra utilizado e ao LEED aplicado, houve uma tendência de diferença significativa, segundo o teste de Levine, com p = 0,084. Conforme o teste, para a fibra linear foi necessária uma LEED média mais alta, de 73,30, enquanto na fibra radial a LEED média foi de 69,21. Quando comparados o fechamento das safenas e a presença de refluxo com o tipo de fibra utilizada, não houve diferença significativa , de acordo com o teste qui-quadrado aplicado.-A IVC pode ser definida como uma alteração do sistema venoso superficial e/ou profundo. A prevalência da doença tem crescido mundialmente e aumenta com a idade. Um estudo feito com uma população de Botucatu, no estado de São Paulo, apontou uma prevalência de varizes de 35,5%laser e os diferentes tipos de fibras ópticasAtualmente, existe um direcionamento dos estudos relacionados ao EVLA em busca de formas mais seguras de utilizá-lo com menos efeitos adversos. Para isso, levam-se em conta dados como o comprimento de onda e a energia utilizada, a velocidade com que se aplica o laser a partir de um gerador levará a uma reação térmica em que a energia do laser é entregue e captada pela água intracelular das células endoteliais da safena. Esse mecanismo de ação pode ser regulado através de parâmetros físicos, tais como comprimento de onda, tipo de administração de energia e quantidade de energia por área de superfície,A liberação do -,-O nível de energia que será absorvido é de extrema importância, uma vez que a retração do vaso só ocorre quando se atingir um determinado patamar; caso contrário, a veia poderá manter-se aberta ou recanalizarO termo LEED começou a ser utilizado em 2005 e tornou-se referência no cálculo da energia utilizada no procedimento devido à sua facilidade e possível padronização. A fórmula que o compõe leva em conta a potência e a velocidade de tração com que se traciona a fibra ópticaNos membros inferiores avaliados neste estudo, a LEED média foi de 70,57 J/cm, e observou-se que, para se obter uma termoablação efetiva na JSF, foi necessária uma LEED acima dessa média. A alta energia proporcionou a obliteração parcialmente completa ou completa na JSF e a ausência de refluxo, resultados também encontrados por Proebstle et al.Em 30 dias de seguimento dos pacientes submetidos a EVLA sob anestesia local para tratamento de IVC, mostrou-se que a utilização de uma LEED acima da média de 70,57 J/cm na JSF teve melhores resultados, com índices maiores de obliteração e ausência de refluxo, em relação a uma LEED menor. No segmento de coxa, não houve diferença significativa. Isso leva à conclusão de que, nesse segmento, não é necessária uma energia mais elevada para que se obtenha o resultado desejado, sendo possível, portanto, utilizar uma LEED mais baixa para diminuir a chance de complicações.Apesar de os resultados serem compatíveis com a literatura, são necessários mais estudos com dados específicos sobre a LEED aplicada na JSF e no segmento de coxa, assim como a sua relação com a incidência de parestesia.


We report the case of a one-year-old girl who was diagnosed with Wiedemann-Steiner Syndrome based on the identification of a novel KMT2A mutations cause Wiedemann-Steiner Syndrome, a very rare genetic disorder characterized by congenital hypertrichosis, short stature, intellectual disability, and distinct facial features. KMT2A; this change generates an alteration of the specific binding to non-methylated CpG motifs of the DNA to the protein. The genotype and phenotype of the patient were compared with those of earlier reported patients in the literature.  Whole exome sequencing identified a novel frameshift variant: c. 4177dupA (p.Ile1393Asnfs * 14) in  In diseases with low frequency, it is necessary to establish a genotype-phenotype correlation that allows the establishment of therapeutic and follow-up goals. The phenotype comparation with other reported cases did not show differences attributable to sex or age among patients with Wiedemann-Steiner Syndrome. Whole exome sequencing allows identifying causality in conditions with high clinical and genetic heterogeneity like hypertrichosis. Non-androgenic excessive growth of terminal hair is associated with several rare genetic conditions et al. de novo dominant mutations in the KMT2A gene  in five of them. They concluded that haploinsufficiency and heterozygous mutations in this gene were the genetic causes of WDSTS. No differences were found for gender. The 49 pathogenic variants reported to date in LOVD 3.0 for the KMT2A gene (https://databases.lovd.nl/shared/variants/KMT2A) correspond mostly to mutations that lead to prematurely truncated proteins ,,KMT2A are associated with defects in chromatin remodeling and, consequently, in the regulation of gene expression ,In 2012, Jones KMT2A. To establish a genotype-phenotype correlation as the major aim of this report, her clinical features are compared with previously reported WDSTS patients.At this stage, the complete phenotype of WDSTS is not understood fully. We report the case of a girl from Colombia with clinical features of WDSTS in whom we identified a not previously reported pathogenic variant of th centile). The baby showed spontaneous neonatal adaptation with APGAR 9 and 10 at 1 and 5 minutes, respectively. She was released jointly with her mother on the second day after birth.We describe the case of a 21-month-old female patient from southwest Colombia, who was the second child of a 34-year-old mother and a nonconsanguineous 36-year-old father, both without a significant family history. The mother’s pregnancy was uncomplicated, and prenatal ultrasounds were normal. A cesarean delivery was performed at 38 weeks because of the breech position of the baby. The birth weight was 3,324 g . At that age, she was also assessed by a pediatric gastroenterologist who diagnosed moderate gastroesophageal reflux that required pharmacological management. Later, at 20 months of age, she presented with two episodes of urinary infection, one of them complicated by pyelonephritis. Regarding her development, she achieved cephalic support at 12 months, and assisted sitting at 18 months. At the age of 21 months, she did not exhibit age-appropriate language development.The paraclinical tests performed on the patient included Normal brain MRI performed at 10 months of age. A renal ultrasound, dimercapto succinic acid renal scan and voiding cystourethrography were performed at 20 months of age and were reported to be normal. Other studies performed at this time were karyotype, blood and urine metabolic screening, creatinine phosphokinase, complete blood count, fasting glucose test, transthoracic echocardiogram, auditory and visual evoked potentials; all of them were reported as normal. X-rays of the extremities performed at 21 months of age showed bilateral congenital hip dislocation.At 21 months of age, she was referred for genetic assessment because of delayed psychomotor development, generalized hypotonia, low height, and hypertrichosis. Her weight was 8,7 kg  and her height was 72 cm . Physical examination revealed round facies, thick eyebrows, synophrys, long eyelashes, downslanted palpebral fissures, hypertelorism, long philtrum, Dennis Morgan folds, and excessive thick facial hair mainly in the frontal region . GeneralKMT2A gene (v1. NM_001197104.1) was identified: c. 4177dupA (p. Ile1393Asnfs*14). This variant was not identified in either of the patient’s parents and has not been reported previously in population databases. This variant generates a change in the reading frame that results in the premature truncation of the protein or degradation of the messenger RNA. This finding was confirmed by Sanger sequencing and was compatible with the diagnosis of WDSTS. No other gene variants were identified in this case.Further investigation was performed using whole exome sequencing (WES) in the trio approach with a massive sequencing platform . WES was performed on the sequencing coding and flanking intronic regions using the HiSeq2500/4000 system . The CASAVA 1.8 analysis package  was used to demultiplex the sequencing reads. The trimmed reads were mapped to the human reference genome (GRCh38) using the Burrows-Wheeler aligner software. A novel frameshift pathogenic variant in the heterozygous state of the http://fathmm.biocompute.org.uk/) and Polymorphism Phenotyping v2 (Polyphen-2 http: // genetics. bwh.harvard.edu/pph2/) classified it as a deleterious/damaging variant (disease causing) because of its high evolutionary conservation.According to the American College of Medical Genetics and Genomics (ACMG) Guidelines for the Interpretation of Sequence Variants de novo inheritance of the mutation was explained to her parents, as was the recurrence risk that varies from 3% to 5% in future pregnancies The The patient’s parents provided written informed consent for the publication of her case report and accompanying images.Exome sequencing has revolutionized the genetic study of monogenic diseases over the last decade. This diagnostic tool allows the time- and cost-effective sequencing of the whole exome (approx. 3% of the genome) to identify genetic causes of dysmorphological syndromes with high clinical and genetic heterogeneity. Exome sequencing has improved diagnostic performance in medical genetic practice by more than 25%, leading to a better understanding of molecular mechanisms involved in pathologies with Mendelian inheritance KMT2A gene have been identified as a cause of this syndrome in studies with large sample sizes that validate the clinical utility of next-generation sequencing tools ,WDSTS is one of the new syndromes identified by exome sequencing since 2012 ,,,,,,It is challenging to diagnose WDSTS because the phenotype shows extensive variation and is not defined clearly. In the young female patient reported here, the facial features appear to be similar to patients reported in other studies. Synophrys, long eyelashes, ocular hypertelorism and long philtrum ,,,Neurological manifestations reported in WDSTS include intellectual disability and psychomotor development delay that are present in 100% of patients, and hypotonia, which can be found in 66.7%. Both of these features were present in our patient. She also exhibited a speech and language developmental delay. Because of her age, an IQ test was not performed. Irritability is a prominent feature in the behavior of WDSTS patients and has been reported in about 20% in combination with hyperactivity, heteroagression, and autistic features KMT2A gene encodes a histone methyltransferase related to the regulation of gene expression. This protein plays an essential role in the DNA packaging for early development and hematopoiesis, mediating chromatin modifications associated with epigenetic transcriptional activation. The KMT2A gene shows broad expression in almost all human tissues ,de novo, meaning a new variant is reported with each clinical report. The majority of mutations are nonsense, frameshift, splice site deletion, or exon deletion, leading to a truncated transcript (messenger RNA) or protein. The mutation reported here c.4177dupA (p.Ile1393Asnfs * 14) is not annotated in the 1,000 Genomes Project, ExAC, or EVSor in-house database, and, so far, has not been reported in the literature. Frameshift type mutations, such as this one, constitute very strong pathogenic criteria according to the ACMG classification of sequencing variants, because they modify the length of the protein by inserting premature stop codons with the shift of the reading frame; therefore, it is usually related to severe phenotypes in monogenic diseases The KMT2A gene that cause WDSTS are distributed throughout the gene, but located before the FYRC domain. This domain is important for the recognition of a protein after cleavage. More than 50% of reported mutations have been in exons 3 and 27, the two longest exons of this gene; however, mutational hot-spot sites have not been identified The pathogenic variants in the KMT2A gene. The phenotype was similar to what is reported in the worldwide literature. This 1-year-old patient had a clinical history of urinary infection episodes without renal impairment or pathological findings in imaging, which seems to be related to the methyltransferase activity of the protein located in the domains and motifs affected by the mutation as described here. In summary, we reported the first case of a patient from Colombia with a frameshift pathogenic variant not reported previously in the This report is an approach to a possible genotype-phenotype correlation for WDSTS, in which, in case of hypertrichosis, neurological and renal involvement must always be ruled out. The most prominent features of WDSTS that can serve as diagnostic criteria are generalized hypertrichosis, postnatal growth retardation, psychomotor development delay and distinct facial phenotype . In more than 70% of the patients reported in the literature, these features are present. ,et al,,,Las bases genéticas de la hipertricosis congénita aún son desconocidas, el crecimiento excesivo del vello terminal no relacionado con andrógenos se asocia a varias condiciones genéticas raros et al.Whole exome secuencing) de pacientes con características clínicas del SWS identificando mutaciones en estado heterocigoto de novo en el gen KMT2A (antes conocido como MLL) en cinco de seis pacientes y concluyendo que la haploinsuficiencia de este gen era la causa genética de esta entidad . Hasta el momento no se han encontrado diferencias en género, las 49 variantes patogénicas reportadas hasta el momento en LOVD 3.0 para el gen (https://databases.lovd.nl/shared/variants/KMT2A) corresponden en su mayoría a mutaciones de novo que generalmente conducen a proteínas truncadas prematuramente ,,KMT2A generan defectos en la remodelación de la cromatina que participa en la regulación de la expresión génica ,En el año 2012, Jones KMT2A, y se comparan sus características clínicas con los pacientes reportados previamente tratando de establecer una correlación genotipo-fenotipo.El fenotipo completo del SWS no se entiende completamente. Se presenta una paciente de origen colombiano con esta entidad, quien además presenta una variante patogénica no reportada en Paciente femenina de 21 meses de edad, producto de la segunda gestación de la madre. La madre con 34 años y el padre con 36 años no consanguíneos. No hay historia familiar significativa. Embarazo normal con ultrasonidos prenatales normales. Parto por cesárea a las 38 semanas de gestación, debido a presentación podálica del feto. Peso al nacer: 3,324 g (Percentil 48), adaptación neonatal espontanea con APGAR 9-10 al 1 y 5 minutos, respectivamente. Fue dada de alta conjunta con su madre al segundo día de nacida.La niña fue valorada al tercer mes de edad por neuropediatría por el cuadro clínico de hipotonía generalizada, asociado a un retraso en el desarrollo psicomotor. Desde los 6 meses de edad se documentaron un bajo peso y talla. Debido a la asociación del cuadro neurológico con hipertricosis, fue valorada a la edad de 12 meses, por endocrinología pediátrica quienes descartaron un trastorno hormonal de tipo androgénico . A esa misma edad fue valorada por gastroenterología y nutrición debido al bajo peso quienes diagnosticaron un reflujo gastroesofágico moderado que requirió un manejo farmacológico. Adicionalmente, a la edad de 20 meses, presentó dos episodios de infección urinaria, uno de ellos complicado con pielonefritis. El desarrollo psicomotor de la paciente fue retrasado, logró el sostén cefálico a los 12 meses, sedestación asistida a los 18 meses y hasta la edad de 21 meses no había un desarrollo del lenguaje. Los paraclínicos realizados incluyen: Resonancia magnética cerebral realizada a los 10 meses de edad con resultado normal. Ecografía renal, DMSA y Cistografía miccional realizadas a los 20 meses, normales. Otros estudios realizados a la edad de 20 meses fueron Cariotipo, tamizaje metabólico en sangre y orina, CPK, hemograma, glicemia en ayunas, ecocardiograma, potenciales evocados auditivos y visuales, todos reportados como normales. Las radiografías de extremidades realizadas a la misma edad evidenciaron luxación congénita bilateral de la cadera.Fue referida para evaluación genética debido a la historia del retraso del desarrollo, hipotonía y baja talla. Al momento de la valoración, a los 21 meses de edad, el peso fue de 8.7 kg (-2.2 DE), altura 72 cm (-3.83 DE) y circunferencia de la cabeza de 47.2 cm (Percentil 30 -0.5 DE). El examen físico evidenció facies redondas, cejas gruesas, sinofris, pestañas largas, fisuras palpebrales inclinadas hacia abajo, hipertelorismo ocular, filtrum largo, pliegues de Dennis Morgan y excesivo vello facial grueso principalmente en región frontal . HipertrWhole exome sequencing (WES) reveló una nueva variante patogénica de novo de tipo frameshift: c. 4177dupA (p. Ile1393Asnfs * 14) en KMT2A (v1. NM_001197104.1), la cual no fue identificada en ninguno de los padres y no se encuentra reportada en las bases de datos poblacionales. Esta variante genera un cambio en el marco de lectura que resulta en el truncamiento prematuro de la proteína o degradación del ARN mensajero, y fue confirmada mediante secuenciación Sanger. Esta variante es compatible con el diagnóstico de Síndrome de Wiedemann-Steiner. No se identificaron más variantes en este estudio. Se realizó el estudio parte de genética de secuenciación exómica utilizando el enfoque de trío . Las regiones codificantes fueron enriquecidas usando la tecnología en solución de Agilent y fueron secuenciadas usando el sistema HiSeq2500/4000 de Illumina. Los datos de salida de Illumina HiSeq se convirtieron de un archivo bcl a un archivo FastQ por el software Illumina CASAVA 1.8, y fueron asignados por el programa Burrows Wheeler Aligner a la secuencia haploide del genoma humano de referencia (GRCh38 https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.38). ACMG variant interpretation guidelinehttp://sift.jcvi.org/), FATHMM (http://fathmm.biocompute.org.uk/) y Polymorphism Phenotyping v2 (Polyphen-2 http://genetics.bwh.harvard.edu/pph2/) la clasifican con efecto deletéreo dada la alta conservación de la posición en vertebrados. Basado en la de novo de la mutación que implica un riesgo de recurrencia que oscila entre el 3 al 5% en otra gestación Se explicó a la familia la herencia Se obtuvo el consentimiento informado de los padres de la paciente para la publicación del reporte de caso y las imágenes acompañantes. . Esta herramienta diagnóstica permite la secuenciación del exoma completo (≈3% del genoma) de manera eficiente en términos de costo y tiempo para la identificación de la causa de síndromes genéticos dismorfológicos con alta heterogeneidad clínica y genética ayudando a comprender los mecanismos moleculares de las patologías con herencia mendeliana La secuenciación del exoma ha revolucionado el estudio genético de enfermedades monogénicas en la última década,KMT2A ha aparecido como un candidato causante de este síndrome en estudios con gran tamaño de muestra que validan la utilidad clínica de la secuenciación de nueva generación,Uno de los síndromes nuevos identificados mediante la secuenciación de exoma es el SWS el cual comparte algunas características clínicas solapantes con otros síndromes que cursan con hipertricosis y que se conoce solo desde el 2012 ,,,,,,Tabla 1.El fenotipo asociado al SWS es amplio por lo que el diagnóstico es un desafío. En la paciente reportada en nuestro reporte de caso, las características faciales parecen ser similares a los pacientes reportados en otros estudios donde se incluye sinofris, pestañas largas, hipertelorismo ocular y filtrum largo ,,,Las manifestaciones neurológicas reportadas en SWS incluyen retraso mental y retraso del desarrollo psicomotor en el 100% de los pacientes e hipotonía en el 66.7%, ambas presentes en la paciente, quien además cursa con retraso del desarrollo del habla. No es posible debido a su edad valorar coeficiente intelectual. El comportamiento se caracteriza por irritabilidad sin ser claro un componente de hiperactividad, lo cual se ha reportado en cerca del 20% de los pacientes con este síndrome, quienes cursan con trastornos del comportamiento como heteroagresión, hiperactividad y rasgos autistas ,de novo, y con cada reporte clínico se reporta también generalmente una nueva variante. La mayoría de las mutaciones son de tipo nonsense, frameshit, splice site o exon deletion las cuales terminan en un transcrito (ARN mensajero) truncado. La mutación aquí reportada c. 4177dupA (p. Ile1393Asnfs * 14) no está presente en 1,000 Genomes Project, ExAC, EVSor in-house database, y no ha sido reportada en la literatura. Las mutaciones de tipo frameshift, como esta constituyen un criterio moderado para la clasificación de patogenicidad de variantes de la ACMG ya que modifican la longitud de la proteína, al insertar codones de parada prematuros con el corrimiento del marco de lectura, por lo que normalmente se relacionan con fenotipos severos en enfermedades monogénicas El gen afectado KMT2A (lisina metiltransferasa 2A) codifica una histona metiltransferasa relacionada con la regulación de la expresión génica, mediante la modificación de la compactación del DNA en el desarrollo temprano y la hematopoyesis en el proceso de regulación de transcripción mediada por la remodelación de cromatina y se expresa ampliamente en la mayoría de los tejidos humanos hot spot mutacional et alLas variantes patogénicas del gen KMT2A causantes del SWS se han distribuida a lo largo de todo el gen, todas localizadas antes del dominio FYRC que es importante para el reconocimiento de la proteína posterior al clivaje. Más de la mitad de las mutaciones se han ubicado en el exón 3 y 27, los cuales son los dos exones más largos del gen, sin embargo, no es claro el comportamiento de sitios En conclusión, se reporta por primera vez una paciente de origen colombiano con una variante patogénica de tipo frameshift no reportada en KMT2A. Sus características son similares fenotípicamente a lo reportado en la literatura, con una historia clínica de episodios de infección urinaria sin alteración renal, lo cual parece relacionarse con la actividad metil transferasa de la proteína ubicada en los dominios afectados por la mutación aquí reportada. Esta sería una aproximación a la correlación genotipo-fenotipo que se propone para el SWS, donde además de la hipertricosis se debe analizar el compromiso neurológico y renal. Las características más prominentes del SWS que pueden servir como criterio diagnóstico son: hipertricosis generalizada, retardo del crecimiento postnatal, retraso del desarrollo psicomotor y fenotipo facial distintivo . Estas características están presentes en más del 70% de los pacientes reportados en la literatura mundial.


To evaluate the magnetic hyperthermia therapy in glioblastoma tumor-on-a-Chip model using a microfluidics device.μL of magnetic nanoparticles (10mgFe/mL) using the parameters that generated the specific absorption rate value. The efficacy of magnetic hyperthermia therapy was evaluated by using the cell viability test through the following fluorescence staining: calcein acetoxymethyl ester (492/513nm), for live cells, and ethidium homodimer-1 (526/619nm) for dead cells dyes.The magnetic nanoparticles coated with aminosilane were used for the therapy of magnetic hyperthermia, being evaluated the specific absorption rate of the magnetic nanoparticles at 300 Gauss and 305kHz. A preculture of C6 cells was performed before the 3D cells culture on the chip. The process of magnetic hyperthermia on the Chip was performed after administration of 20Magnetic nanoparticles when submitted to the alternating magnetic field (300 Gauss and 305kHz) produced a mean value of the specific absorption rate of 115.4±6.0W/g. The 3D culture of C6 cells evaluated by light field microscopy imaging showed the proliferation and morphology of the cells prior to the application of magnetic hyperthermia therapy. Fluorescence images showed decreased viability of cultured cells in organ-on-a-Chip by 20% and 100% after 10 and 30 minutes of the magnetic hyperthermia therapy application respectively.The study showed that the therapeutic process of magnetic hyperthermia in the glioblastoma on-a-chip model was effective to produce the total cell lise after 30 minutes of therapy. As a result of the studies developed in this area, a number of products have been commercialized and used in clinical medicine, such as biocompatible nanoparticles applied both to therapy and to diagnosis of tumors. One of the techniques applied to treatment of tumors is hyperthermia that uses strategies based on radiofrequency waves, ultrasound and microwave, as well as laser light treatments associated with the use of nanoparticles with magnetic properties.(–) Magnetic nanoparticles (MNP), when submitted to alternating magnetic field (AMF), generate heat by transforming magnetic energy into thermal energy used in treatment of tumors, such as shown in In the last decade, nanotechnology and nanomaterials has led to advances and introduced a new area in medicine, the nanomedicine. consist of administration of MNP in tumor tissue and heating of tumor cells to temperature between 41 to 43°C, by application of AMF, depending on magnetic field (B) and frequency of oscillation (freq) as shown in Treatment of GBM, using the MHT technique, The assessment of new therapeutic approach for treatment of GBM by using MHT technique was showed in vitro and murine models, however, this in vitro models did not respond clearly the reason why they are incapable of mimic tumor in an environment that the tumor is, and, in murine models, there are a difference between use xerographic or autologous models. However, in 3D cell culture model, which use technology of microfluidics devices (chip), cultivated tumor cells mimic the development of tumor tissue most near to reality, enabling, in this system, the assessment of therapeutic approaches, such as MHT, which does not occur in model in vitro 2D, which is not so efficient to visualize the behavior of cancer cells during coculture.Glioblastoma tumors studies with MHT technique have been emphasized because they increase patient's survival compared with other treatments, in addition to not present relevant collateral effects.By using organ-on-a-chip modality, it is possible to mimic tumors of GBM and apply therapy of MHT after administration of dispersed MNP in aqueous medium in cavities of chip, such as shown in To evaluate efficiency of magnetic hyperthermia therapy in glioblastoma on-a-chip.Hospital Israelita Albert Einstein, Sao Paulo, Brazil.The study was carried out in an Experimental Research Center and Experimental and Surgical Training Center of the Teaching and Research Institute at 3O4), being coated with aminosilane, which turns it biocompatible. The hydrodynamic diameter of MNP is 100nm with number of nanoparticles ~1.8×1015/g and density of ~1.25g/cm3.Dispersed MNP in aqueous medium forming one ferrofluid, available commercially as fluidMAG-Amine , have used in therapy of MHT in GBM on-a-chip model. The MNP has nucleus with crystalline phase of magnetite  of adjustable magnetic field (50-300 Gauss) in a variety of modes of frequencies ; and controller module DMC1 , which enabled to conduct program of trials, monitoring of measures and analysis of results. The monitoring of temperature was taken using fiber optic temperature measurement sensors . The system was controlled by software MaNiaC , which enabled program and data processing.Determination of specific absorption rate (SAR) of MNP (10mgFe/mL) was conducted by submitting these to AMF , registering ranging temperature over time that, for statistical purposes, four measurements were done. Calculations of SAR was done using MaNiaC software, using the relationmNP is the mass of MNP, m1 the mass of water (1000kg/m3), cNP the specific heat of magnetite , c1 the specific heat of water  and (dT/dt)max is maximal ranging of heating curve of MNP.in which in vivo tumors and share a number of malignant characteristics with GBM human.In this study, we used C6 cells of glioma, a GBM lineage multiform of rats , which has the ability to form ® Invitrogen Corporation, CA, USA), supplemented with 10% of fetal bovine serum (FBS) , 1% of penicillin- streptomycin  and 1% of L-glutamine  at 37°C (5% CO2), in bottles of cell culture of 75cm2 . When achieve cell confluence of 85%, cells were trypsinized using 0.25% trypsin , collected, centrifuged at 800rpm for 5 minutes, resuspended in culture medium in cell concentration of 107cells/mL and keep refrigerated on ice bath.These C6 cells were cultivated using RPMI   were injected in central compartment, using a syringe and sterile Tygon tubing (0.02″ ID × 0.06″ OD) SynVivo Inc, Huntsville, AL, US) and kept on the fridge at 5°C for a period of 2 hours. Culture medium non-supplemented RPMI was injected for washing compartment, and C6 cells (107cells/mL), with flow rate of 2μL/minute, using bomb 11 Elite Nanomite . External compartment was supplemented RPMI with 10% SFB to 5μL/minute and maintained during all period of culture. The chip was placed in 5% carbon dioxide oven and 37°C for 3D cell culture of C6 cells, with change of culture mean of cavity every 4 hours, during 48 hours.To mimic tumor tissue of GBM, we used microfluidic device from SynVivo Inc . This chip was formed by two compartments, one central and the other external, separated by porous interface, aiming the culture of the C6 cells in 3D in central cavity, being prepared as described in SynVivo protocol.μL of MNP, in concentration of 10mgFe/mL in the same local using infusion pump. The chip was taken up to the MHT equipment and placed on applicator, as shown in μL contained in eppendorf in concentration of 10mgFe/mL) as referential sample that was submitted to same AMF together with chip and the temperature was monitored by fiber optic system.After 48 hours of 3D cell culture of C6 cells on-a-chip, we performed the MHT assay. For this reason, the chip was removed from the incubator, and central cavity was washed with RPMI (0%FBS); subsequently, we injected 20® kit  of assay of viability and/or cell toxicity, through image of fluorescence, in which were used 4mM of Calcein acetoxymethyl ester (Ca-AM) and 12mM of ethidium homodimer-1 (EthD-1). The fluorescence of both staining occur to interact with live cells (for Ca-AM-excitation/emission: 492/513nm) or dead cells (for EthD-1 – excitation/emission: 526/619nm). Green fluorescence of acetoxymethyl ester indicates the activity of intracellular esterases of viable cells, and red fluorescence of EthD-1 indicates loss of integrity of plasmatic membrane. The analysis of cell viability before and after the MHT therapy in tumor cells on-a-chip was conducted by injecting 15μL of solution formed by Ca-AM and EthD-1 in central cavity of chip and, subsequently, we registered fluorescence images, using inverted microscopy Nikon Eclipse Ti-E . The counting of live (green) and dead (red) cells were done in two regions of organ-on-a-chip . The experiment since the 3D cell cultivation up to assessment of cell viability was repeated three times.The assessment of efficiency of therapy of MHT in GBM on-a-chip was conducted using LIVE/DEADCapacity of heating of MNP was characterized for application in MHT. The heating curve of MNP is shown in 3D cell culture of C6 cells was conducted in central cavity of chip and evaluated through images of microscopy of clear field. ® structure mimicking the formation of GBM tumor tissue.Images of After growth of tumor tissue on the chip, we applied the therapy of MHT to conduct assay of viability of C6 cells, such as shown in ®).Fluorescence assay of in vitro studies. The development of these microfluidic systems to mimic tumors are on use in a number of therapies, raising the interest of scientific community, in order to replace the use of murine models.(–) One of these therapies is applied by hyperthermia treatments, such as MHT in tumors.Microfluidics have provided a large development in tissue engineering, aiming to understand biologic processes  This can be observed in review of Gupta et al., which describe different parameters of applications of MHT in models of tumor in vitro and characteristics of MNP used. In the study by Hanini et al., an evaluation was conducted of MHT in glioma cells (U87-MG) treated with MNP of γ-Fe2O3 coated with polyol, with diameter of 10nm, in concentration of 50μgFe/mL, submitted to AMF with frequency of oscillation of 700kHz and magnetic field of 289.67Oe, with time of application of 60 minutes, keeping the therapeutic temperature of 42°C and showing reduction cell viability of 50%. In other study, we used cell of glioma T-9 and MNP of magnetite with diameter of 35nm in concentration of 7.2mgFe/mL, applying AMF with 118kHz and 383.72Oe, achieving cell lyse of 100% in 60 minutes. By using the same tumor cell used in our study, Gupta et al., evaluated C6 glioma cells of rats and NIH3T3 fibroblast of mice, using MNP of Fe2O3 coated with steviosides, of 4.62nm of diameter, in concentration of 100μgFe/mL, applying AMF with 405kHz and 168Oe for 30 minutes, achieving therapeutic temperature of 43°C, and showing a decrease in cell viability of 40% and, after 4 hours of coculture, an additional reduction of 34%. The enhancement of nanomaterial for this therapeutic approach has also been the focus of this study to obtain the best SAR that reflects in efficiency of MHT technique.Studies of MHT in tumor cells using MNP show a large potential in treatment for tumors of GBM, however, because of variety of parameters of application of MHT and the use of different types of tumor cells, it has been difficult to evaluate which are the best parameters of this therapy in tumor treatment, as well as this constitutes a barrier for application of this modality as standard in treatment of GBM.in vivo studies, such the one conducted by Jordan et al., the efficacy of therapy of MHT was evaluated in brain tumor of Fisher's rats induced by RG-2 cells, with two types of MNP - one coated with aminosilane and other dextran. Results showed that application of AMF (100kHz and 225.72Oe) with MNP coated with aminosilane was more efficient in reduction of rate of cell proliferation than when coated with dextran. This study had adequate values of SAR, on the range of 10 to 100W/g – values considered typical of SAR for this type of application. In our study, the MNP of iron oxide (magnetite) were also coated with aminosilane and mean value of SAR of these MNP was 115.4±6.0W/g. Clinical studies published in the literature have also reported(–) the used of this type of MNP coated with aminosilane, and parameters of application of MHT are similar to those used in our GBM study on-a-chip. These similar characteristic, also use the C6 tumor cells that mimic the human GBM, assist in transposition of data for human model, allowing better evaluation of altered therapeutic approach, such as the MHT, combined or not with other techniques in high-severity illness and low response to conventional treatments such as the GBM.However,  screening of high performance drugs and prolonged drug delivery;(–) to evaluate vascular compartment that present a network of vessels in communication with solid 3D tumors mimicking microenvironment of tumor, including the knowledge as Enhanced Permeability and Retention (EPR), among others.Currently, the organ on-a-chip model of GBM has been used to evaluate the ability to model progression of hyper cellular regions of GBM, observed in patients, and mimicking the obstruction of blood vessels, modeling the delivery of nutrients, and gradients of oxygen during the evolution of GBM;For this reason, the optimization of therapy of MHT in microfluidic device that mimic the characteristics of GBM is presented as potential application for translational in humans.The models of GBM on-a-chip of our study provides basis for implementation of this method technique of MHT, aiming to evaluate its potential therapeutic in GBM, temporally, although our model has presented one limiting factor, which was the lack of vascular network associated with tumor tissue, but this must be implemented in future studies.Our study showed efficiency of magnetic hyperthermia therapy for treatment of glioblastoma on-a-chip with lise of all tumor cells after 30 minutes of magnetic hyperthermia using nanoparticles of iron oxide coated with aminosilane, which is used in clinical trials. In addition, specific absorption rate was often used in therapy assays of magnetic hyperthermia in tumors of human glioblastoma.  Como resultado das pesquisas desenvolvidas nesta área, diversos produtos já estão sendo comercializados e usados na clínica médica, como as nanopartículas biocompatíveis aplicadas tanto na terapia como no diagnóstico de tumores. Uma das técnicas aplicadas no tratamento de tumores é a terapia hipertérmica, que utiliza estratégias baseadas em ondas de radiofrequência, ultrassom e microndas, bem como tratamentos com laser associado ao uso de nanopartículas com propriedades magnéticas.(–) As nanopartículas magnéticas (NPM), quando submetidas a um campo magnético alternado (CMA), geram aquecimento, mediante a transformação de energia magnética em energia térmica usada no tratamento de tumores, como mostrado na Na última década, a nanotecnologia e os nanomateriais deram lugar a uma nova área na medicina, a denominada “nanomedicina”. consiste na administração de NPM no tecido tumoral e no aquecimento das células tumorais a uma temperatura entre 41 a 43°C, mediante aplicação do CMA, dependente do campo magnético (B) e frequência de oscilação (freq) como é mostrado na O tratamento do GBM, mediante a técnica de MHT,Estudos de GBM com a técnica da MHT vêm se destacando por apresentarem aumento da sobrevida do paciente quando comparado a outros tratamentos, além de não apresentar efeitos colaterais relevantes.in vitro e murinos, mas os modelos in vitro não respondem de forma clara, porque não são capazes de mimetizar o tumor no ambiente no qual ele se encontra, e, nos modelos murinos, existe uma diferença entre usar modelos xenográficos ou autólogos. Entretanto, no modelo de cultura celular 3D, que utiliza a tecnologia dos dispositivos microfluídicos (chip), as células tumorais cultivadas mimetizam o desenvolvimento do tecido tumoral de maneira mais próxima do real, possibilitando, nesse sistema, a avaliação de abordagens terapêuticas, como da MHT, o que não acontece no modelo 2D in vitro, o qual não se mostra tão eficiente para visualizar o comportamento de células cancerígenas durante a cocultura.A avaliação de novas abordagens terapêuticas visando ao tratamento do GBM por meio da técnica da MHT foi demonstrada nos modelos organ-on-a-chip, é possível mimetizar tumores de GBM e aplicação da terapia da MHT após a administração das NPM dispersas no meio aquoso nas cavidades do chip, como mostrado na on-a-chip, como canais adequados para o desenvolvimento do tecido tumoral, correta administração de drogas/nanopartículas, troca de fluidos para a manutenção celular, entre outras  reduzindo o uso de animais.Com o uso da modalidade e outras . Outra von-a-chip.Avaliar a eficiência da terapia de magneto-hipertermia em tumores de glioblastoma O estudo foi realizado no Centro de Pesquisa Experimental e no Centro de Experimentação e Treinamento em Cirurgia do Instituto Israelita de Ensino e Pesquisa do Hospital Albert Einstein, São Paulo, Brasil.fluidMAG-Amine , foram usadas na terapia da MHT no modelo GBM on-a-chip. As NPM possuem núcleo com fase cristalina da magnetita (Fe3O4), sendo cobertas com aminosilana, que as torna biocompatíveis. O diâmetro hidrodinâmico das NPM é de 100nm, com número de nanopartículas ~1,8×1015/g e densidade de ~1,25g/cm3.As NPM dispersas em meio aquoso formando um ferrofluido, disponíveis comercialmente como A terapia de MHT no dispositivo microfluídico foi aplicada usando sistema de aquecimento magnético composto por: aplicador DM100  de campo magnético ajustável (50-300 Gauss) em diversos modos de frequências ; e um módulo controlador DMC1 , que permitiu realizar a programação dos ensaios, monitorização das medições e análise dos resultados. A monitorização da temperatura foi realizada mediante sonda de temperatura de fibra ótica . O sistema foi controlado pelo software MaNiaC , que facilitou a programação e o processamento de dados.specific absorption rate) das NPM (10mgFe/mL) foi realizada submetendo estas ao CMA , registrando a variação da temperatura no tempo e que, para fins estatísticos, foram realizadas em quatro medidas. O cálculo da SAR foi realizado mediante o software MaNiaC, utilizando a relaçãoA determinação da taxa de absorção específica , NPc o calor específico da magnetita , 1c o calor específico da água  e (dT/dt)max é máxima variação da curva de aquecimento das NPM.onde in vivo e compartilhar várias características malignas com o GBM humano. Estas células C6 foram cultivadas em meio RPMI , suplementado com 10% de soro fetal bovino (SFB) , 1% de penicilina-estreptomicina  e 1% de L-glutamina  a 37°C (5% CO2), em garrafas de cultura celular de 75cm2 . Ao atingir confluência celular de 85%, as células foram tripsinizadas utilizando 0,25% tripsina , coletadas, centrifugadas a 800rpm por 5 minutos, ressuspensas em meio de cultura na concentração celular de 107 células/mL e mantidas resfriadas em banho de gelo.Foram utilizadas, neste estudo, células C6 de glioma, uma linhagem de GBM multiforme de ratos , que tem a capacidade de formar tumores chip era formado por dois compartimentos, um central e outro externo, separados por interface porosa, visando à cultura de células C6 em 3D na cavidade central, sendo preparado como descrito no protocolo da SynVivo. Basicamente, 15μL de Matrigel® (40mg/cm2)  foram injetados no compartimento central, utilizando seringa e tubulação estéril de Tygon   e mantidos na geladeira a 5oC por um período de 2 horas. Meio de cultura RPMI não suplementado foi injetado para lavagem do compartimento, e as células C6 (107celúlas/mL) foram semeadas na cavidade central, a uma vazão de 2μL/minuto, utilizando bomba 11 Elite Nanomite . No compartimento externo, foi escoado RPMI suplementado com 10% SFB a 5μL/minuto e mantido durante todo o período de cultura. O chip foi colocado em estufa de dióxido de carbono a 5% e 37°C para a cultura 3D das células C6, com troca do meio de cultura da cavidade a cada 4 horas, durante 48 horas.Para mimetizar o tecido tumoral de GBM, foi utilizada o dispositivo microfluídico da SynVivo Inc . Este on-a-chip, foi realizado o ensaio de MHT. Para tal finalidade, o chip foi retirado da estufa, e a cavidade central foi lavada com RPMI (0%SFB); seguidamente, foram injetados 20μL de NPM, na concentração de 10mgFe/mL no mesmo local, utilizando a bomba de infusão. O chip foi levado ao equipamento de MHT e colocado no aplicador, como mostrado na chip por um período de 30 minutos. Com a finalidade de ter o controle da temperatura na faixa terapêutica de 41 a 43oC, foi utilizada quantidade da suspensão coloidal de NPM (200μL contidos em um eppendorf na concentração de 10mgFe/mL) como amostra referencial, que foi submetida ao mesmo CMA, em conjunto com o chip e a temperatura monitorada mediante o sistema de fibra ótica.Após 48 horas de cultura 3D das células C6 on-a-chip foi realizada usando o kit LIVE/DEAD®  de ensaio de viabilidade e/ou toxicidade celular, mediante imagem de fluorescência, no qual foram usados 4mM de acetoximetiléster de calceína (Ca-AM) e 12mM de etídio homodímero-1 (EthD-1). A fluorescência de ambos os corantes ocorre ao interagir com células vivas (para Ca-AM – excitação/emissão: 492/513nm) ou mortas (para EthD-1 – excitação/emissão: 526/619nm). O verde fluorescente da acetoximetiléster indica a atividade de esterase intracelular de células viáveis, e o vermelho fluorescente do EthD-1 indica perda de integridade da membrana plasmática. A análise da viabilidade celular antes e após a terapia de MHT nas células tumorais on-a-chip foi realizada injetando 15μL da solução formada por Ca-AM e EthD-1 na cavidade central do chip e, seguidamente, foram registradas imagens de fluorescência, utilizando um microscópio invertido Nikon Eclipse Ti-E . As contagens de células vivas (verde) e mortas (vermelhas) foram feitas nas duas regiões do organ-on-a-chip . O experimento desde o cultivo celular 3D até a avaliação da viabilidade celular foi repetido três vezes.A avaliação da eficiência da terapia de MHT no GBM inset da box plot) mostra a avalição da distribuição dos valores da SAR com valor médio de 115,4±6,0W/g.A capacidade de aquecimento das NPM foi caracterizada para aplicação na terapia da MHT. A curva de aquecimento das NPM é mostrada na chip e avaliada mediante imagens de microscopia de campo claro. A chip.A cultura 3D das células C6 foi realizada na cavidade central do ® mimetizando a formação de um tecido tumoral de GBM.Nas imagens da chip, foi aplicada a terapia de MHT para realização do ensaio de viabilidade das células C6, como mostrado na Chip, respectivamente. Na on-a-chip. Na Após crescimento do tecido tumoral no kit de viabilidade celular (LIVE/DEAD®).Os ensaios de fluorescência da in vitro. O desenvolvimento destes sistemas microfluídicos para mimetizar tumores está sendo utilizado em diversas terapias, despertando o interesse na comunidade científica, afim de substituir o uso de modelos murinos.(–) Uma destas terapias é a aplicação de tratamentos hipertérmicos, como o MHT em tumores.A microfluídica tem proporcionado grande desenvolvimento na engenharia de tecidos, visando ao entendimento de processos biológicos em estudos  Isto pode ser verificado na revisão de Gupta et al., que descreveram os diferentes parâmetros de aplicação do MHT em modelos de tumor in vitro e as características da NPM utilizadas. No estudo de Hanini et al., foi realizada a avaliação do MHT em células de glioma (U87-MG) tratadas com NPM de γ-Fe2O3 recobertas com poliol, com diâmetro de 10nm, na concentração de 50μgFe/mL, submetidas ao CMA com frequência de oscilação de 700kHz e campo magnético de 289,67Oe, com tempo de aplicação de 60 minutos mantendo-se a uma temperatura terapêutica de 42°C e mostrando diminuição na viabilidade celular de 50%. Em outro estudo, foram utlizadas células de glioma T-9 e NPM à base de magnetita com diâmetro de 35nm na concentração de 7,2mgFe/mL, aplicando CMA com 118kHz e 383,72Oe, alcançando lise celular de 100%, em 60 mimutos. Já com a mesma célula tumoral utilizada em nosso estudo, Gupta et al., avaliaram as células de glioma C6 de rato e fibroblasto de camundongo NIH3T3, utilizando NPM de Fe2O3 revestidas com esteviosídeo, de 4,62nm de diâmetro, na concentração de 100μgFe/mL, aplicando CMA com 405kHz e 168Oe durante 30 minutos, alcançando temperatura terapêutica de 43°C, e mostrando decréscimo na viabilidade celular de 40% e, após 4 horas de cocultura, diminuição adicional de 34%. O aprimoramento dos nanomateriais para esta abordagem terapêutica também tem sido foco de estudo para a obtenção da melhor SAR, que reflete na eficiência da técnica de MHT.Estudos de MHT em células tumorais utilizando NPM mostram grande potencial no tratamento para tumores de GBM, porém, devido à variedade de parâmetros da aplicação de MHT e ao uso de diferentes tipos de células tumorais, tem sido difícil a avaliação de quais são os melhores parâmetros desta terapêutica no tratamento tumoral, bem como configura-se um obstáculo para aplicação desta modalidade como padrão no tratamento de GBM.in vivo, como de Jordan et al., foi avaliada a eficácia da terapia da MHT, em tumor cerebral de ratos Fisher induzido com células RG-2, com dois tipos de NPM, uma recoberta com aminosilana e outra com dextrana, mostrando que a aplicação do CMA  com NPM recoberta com aminosilana foi mais eficiente na diminuição da taxa de proliferação celular do que quando revestidas com dextrana. Este estudo apresentou valores de SAR adequada, na faixa de 10 a 100W/g – valores considerados típicos de SAR para este tipo de aplicação. Em nosso estudo, as NPM de óxido de ferro (magnetita) também foram recobertas com aminosilana e o valor médio de SAR destas NPM foi de 115,4±6,0W/g. Estudos clínicos reportados na literatura(–) também têm usado este tipo de NPM recoberto com aminosilana, e os parâmetros de aplicação de MHT são semelhantes aos usados em nosso estudo de GBM on-a-chip. Estas características similares, como também a utilização de células tumorais C6 que mimetizam o GBM humano, auxiliam na transposição dos dados para o modelo humano, permitindo melhor avaliação de abordagens terapêuticas alternativas, como o MHT, combinadas ou não com outras técnicas em doenças com alta severidade e baixa resposta aos tratamentos convencionais como são as GBM.Já em estudos organ on-a-chip de GBM tem sido utilizado para avaliar a capacidade de modelar a progressão de regiões hipercelulares do GBM, observada em pacientes, imitando a obstrução de vasos sanguíneos e modelando a entrega de nutrientes e gradientes de oxigênio durante a evolução do GBM; triagem de drogas de alto rendimento e liberação prolongada de drogas;(–) avaliar o compartimento vascular que apresenta uma rede de vasos em comunicação com tumores sólidos 3D imitando microambiente do tumor, incluindo o conhecido como efeito Enhanced Permeability and Retention (EPR), entre outros. Portanto, a optimização da terapia da MHT em um dispositivo microfluídico que mimetize as características do GBM se apresenta com potencial aplicação para o translacional em humanos.Atualmente, o modelo on-a-chip do presente trabalho proporcionou bases para a implementação da metodologia da técnica de MHT, visando avaliar seu potencial terapêutico em GBM temporalmente, embora nosso modelo tenha apresentado um fator limitante, que foi a falta da rede vascular associada ao tecido tumoral, mas que deve ser implementada em trabalhos futuros.O modelo de GBM on-a-chip, com lise de todas as células tumorais após 30 minutos de terapia de magneto-hipertermia, usando nanopartículas de óxido de ferro recobertas com aminosilana (usadas em ensaios clínicos). Também, o valor de taxa de absorção específica foi o tipicamente usado em ensaios de terapia de magneto-hipertermia em tumores de glioblastoma humano.O presente estudo mostrou eficiência da terapia de magneto-hipertermia no tratamento de tumor de glioblastoma


There is an error in affiliation 1 for authors Anne Guhn, Angela Merkl, Philipp Sterzer, and Stephan Köhler. The correct affiliation 1 is: Charité –Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Psychiatry and Psychotherapy, Campus Mitte, Berlin, Germany.The following information is missing from the Funding statement: The authors acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charité –Universitätsmedizin Berlin.