CRISPR/Cas9-Directed Gene Trap Constitutes a Selection System for Corrected BCR/ABL Leukemic Cells in CML.

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

Vitamin D concentration and its association with parathyroid hormone in children and adolescents / Concentración de vitamina D y su relación con la hormona paratiroidea en niños y adolescentes

Abstract Background: Vitamin D (VD) deficiency has been inversely associated with parathyroid hormone (PTH) levels in the adult population but not in children and adolescents. This study aimed to report VD concentration and its correlation with PTH levels in a sample of healthy Mexican children. Methods: We conducted a cross-sectional study with 275 healthy Mexican subjects aged 2 to 17 years to estimate the status of 25-(OH)-D and its correlation with PTH levels. The 25-(OH)-D levels were estimated by liquid chromatography-tandem mass spectrometry and PTH by radioimmunoassay. Results: Subjects were categorized as young children (2 to 5 years), school children (6 to 10 years), and adolescents (11 to 17 years). The median concentration of 25-(OH)-D in young children was 27.4 ng/ml; in school children, 25.6 ng/ml; and adolescents, 24.7 ng/ml. VD levels < 20 ng/ml were found in only 10.5% of the participants. Only 3% of the young children showed VD deficiency, in contrast to 10% of school children and 21% of adolescents (p ≤ 0.05). PTH was found within normal ranges in 95.6% of the studied population. VD levels < 20 mg/dl were found in 25.8% of children with overweight or obesity (p = 0.009). Conclusions: VD levels < 20 ng/ml were observed in 10% of the studied group, but this percentage increased with age: 21% of the adolescents showed VD levels < 20 ng/ml. No correlation with PTH levels was found. The VD values reported in this study are lower than those previously reported in Mexican children.

Resumen Introducción: La deficiencia de vitamina D se ha asociado inversamente con la concentración de hormona paratiroidea (PTH) en los adultos, pero no en los niños y adolescentes. El objetivo de este estudio fue reportar la concentración de vitamina D y su correlación con la concentración de PTH en una muestra de niños mexicanos sanos. Métodos: Se llevó a cabo un estudio transversal con 275 mexicanos sanos de 2 a 17 años de edad en quienes se estimaron las concentraciones de 25-(OH)-D utilizando cromatografía líquida con espectrometría de masas y de PTH por radioinmunoensayo, y su correlación. Resultados: Los participantes se categorizaron como prescolares (2 a 5 años), escolares (6 a 10 años) y adolescentes (11 a 17 años). La mediana de la concentración de 25-(OH)-D en los prescolares fue de 27.4 ng/ml, en los escolares de 25.6 ng/ml y en los adolescentes de 24.7 ng/ml. Solo en el 10.5% de los participantes se encontraron valores de vitamina D < 20 ng/ml. En contraste con el 10% de los escolares y el 21% de los adolescentes, solo el 3% de los prescolares mostraron deficiencia de vitamina D (p ≤ 0.05). La PTH se encontró dentro de los límites normales en el 95.6% de la población estudiada. Se encontraron concentraciones de vitamina D < 20 mg/dl en el 25.8% de los niños con sobrepeso y obesidad (p = 0.009). Conclusiones: Solo en el 10% de los participantes se encontraron concentraciones de vitamina D < 20 ng/ml, pero este porcentaje aumento con la edad y se hallaron valores < 20 ng/ml en el 21% de los adolescentes. La PTH se encontró dentro de los valores normales. Las concentraciones de vitamina D en este estudio resultaron menores que las previamente reportadas en niños mexicanos.

Vitamin D, not iron, is the main nutrient deficiency in pre-school and school-aged children in Mexico City: a cross-sectional study.

INTRODUCTION: In 2012, the Mexican National Health Survey showed a moderate prevalence rate of vitamin D deficiency, around 16%, in a national representative sample of children. A decreasing prevalence of anemia during the last 15 years has been observed in Mexico. The aim of this study was to determine the levels of vitamin D in children 3-8 years old in four different locations within the metropolitan area of Mexico City and to compare them to levels of iron and zinc as references of nutritional status. METHODS: One hundred and seventeen healthy children aged 3-8 years attending four hospitals in Mexico City were invited to participate. All children received medical and nutritional evaluation, and blood samples were obtained. RESULTS: Children were selected in the four hospitals between April and August 2008. More than half (51.3%) were boys; their average age was 5.5 ± 1.6 years. The mean height and weight of the children were 112.1 ± 11.2 cm and 20.2 ± 4.9 kg respectively, with a body mass index [BMI] of 15.8 ± 1.7 kg/m². The mean Z-score (BMI) was 0.007 ± 0.999. The prevalence of subjects with deficient levels of 25-OH-vitamin D (<50 nmol/l) was 24.77%. None of the children had haemoglobin levels below the anaemia threshold, and zinc determination revealed 8.26% of individuals with deficient levels (<65 µg/dL). These data confirm the findings reported in the latest National Nutrition Survey (ENSANUT 2012) about the sustained reduction of anaemia prevalence among preschool and schoolchildren since 1999 and the rising rates of vitamin D deficiency in the same population. Similar to other studies, we found a link between socioeconomic status and the deficiency of micronutrients, these being markers of better nutrition, and vitamin D is remarkably related to the quality of the diet. This finding has not been considered in our population before. CONCLUSIONS: There is evidence of a sustained decrease of anaemia in Mexican children due to general enrichment of foods and focus on vulnerable populations, while vitamin D deficiency seems to have increased. More studies are needed to obtain more information on vitamin D levels at different ages and definition of susceptible groups in order to investigate the possibility of general population measures such as enrichment, which have proven to be effective.

Deficiencia de vitamina D en la edad pediátrica. Una oportunidad de prevención / Vitamin D deficiency in childhood: an opportunity for prevention

ResumenLa prevalencia de deficiencia de vitamina D en la población pediátrica ha incrementado en los últimos años y se considera que continúa subdiagnosticada y subtratada. De acuerdo con datos de la Encuesta Nacional de Salud y Nutrición 2006, en México se ha estimado una prevalencia del 16% en niños de 2 a 12 años. La vitamina D desempeña un papel fundamental en la formación y homeostasis del hueso, y consecuentemente en el crecimiento. Su deficiencia se asocia con enfermedades como raquitismo y osteomalacia, y se ha relacionado con otros padecimientos, como obesidad, síndrome metabólico, diabetes, cáncer, infecciones de vías respiratorias y problemas del sistema inmune. En la literatura se han descrito grupos específicos de riesgo para deficiencia de vitamina D en los que el suplemento pudiera ofrecer un beneficio. Actualmente aún hay controversia en definir los niveles séricos de suficiencia, así como la dosis de suplemento. En México, la ingesta diaria sugerida de vitamina D es de 5.6 µg/día (224 UI), que resulta significativamente menor a las recomendaciones en los Estados Unidos y Europa (entre 400 y 1,000 UI). Debido al aumento en la deficiencia de vitamina D en los últimos años y a la falta de consenso con respecto a los niveles de suficiencia de vitamina D (ya que los valores de corte varían de 20 a 30 ng/ml considerados por la asociación de endocrinología), el objetivo de esta revisión fue proporcionar un panorama general del problema en la población pediátrica, así como describir aquellos grupos en riesgo y analizar las recomendaciones vigentes para el suplemento de vitamina D.La deficiencia de vitamina D se ha considerado rara en México, y la falta de evidencia no ha permitido establecer las recomendaciones de ingesta diaria, de acuerdo con el Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. Sin embargo, hoy debe reconocerse como un problema de salud, meritorio de atención y acción. Sugerimos que se lleven a cabo estudios prospectivos en nuestro país, donde se establezca la relación entre la deficiencia sérica de vitamina D y la pobre mineralización ósea.

AbstractThe prevalence of vitamin D deficiency in the pediatric population has increased in recent years and continues to be underdiagnosed and undertreated. According to data from the "ENSANUT 2006" (National Health and Nutrition Survey), the prevalence of vitamin D deficiency in Mexico was 16% in children aged 2-12 years. Vitamin D plays a critical role in the formation and bone homeostasis and consequently on growth. Its deficiency is clearly associated with diseases such as rickets and osteomalacia, and it has been linked to other diseases such as obesity, metabolic syndrome, diabetes, cancer, respiratory infections and immune system disease. Specific risk groups have been described in the medical literature for vitamin D deficiency in which supplementation may offer a benefit. Currently, there is still controversy in defining the serum levels of proficiency and dose supplementation. In Mexico, the daily suggested intake of vitamin D is 5.6 µg (224 IU), which is significantly lower than the recommendations in the U.S. and Europe (i.e., between 400 and 1000 IU/day).An increase in vitamin D deficiency has been reported in recent years. There is no consensus regarding the sufficiency levels of vitamin D. Cut-off values vary from 20 to 30 ng/ml. Therefore, the objective of this review was to provide an overview of the problem in the pediatric population and to describe the groups at risk, as well as to analyze the current recommendations for vitamin D supplementation.Vitamin D deficiency was considered rare in Mexico according to the National Institute of Medical Science and Nutrition Salvador Zubirán. Lack of evidence did not help to establish the international recommended daily intake. Currently, vitamin D deficiency must be recognized as a health problem, worthy of attention and action. We suggest that prospective studies are carried out in our country where the relationship between serum vitamin D deficiency and poor bone mineralization will be established.

[Vitamin D deficiency in childhood: an opportunity for prevention]. / Deficiencia de vitamina D en la edad pediátrica. Una oportunidad de prevención.

The prevalence of vitamin D deficiency in the pediatric population has increased in recent years and continues to be underdiagnosed and undertreated. According to data from the "ENSANUT 2006" (National Health and Nutrition Survey), the prevalence of vitamin D deficiency in Mexico was 16% in children aged 2-12 years. Vitamin D plays a critical role in the formation and bone homeostasis and consequently on growth. Its deficiency is clearly associated with diseases such as rickets and osteomalacia, and it has been linked to other diseases such as obesity, metabolic syndrome, diabetes, cancer, respiratory infections and immune system disease. Specific risk groups have been described in the medical literature for vitamin D deficiency in which supplementation may offer a benefit. Currently, there is still controversy in defining the serum levels of proficiency and dose supplementation. In Mexico, the daily suggested intake of vitamin D is 5.6µg (224 IU), which is significantly lower than the recommendations in the U.S. and Europe (i.e., between 400 and 1000 IU/day). An increase in vitamin D deficiency has been reported in recent years. There is no consensus regarding the sufficiency levels of vitamin D. Cut-off values vary from 20 to 30ng/ml. Therefore, the objective of this review was to provide an overview of the problem in the pediatric population and to describe the groups at risk, as well as to analyze the current recommendations for vitamin D supplementation. Vitamin D deficiency was considered rare in Mexico according to the National Institute of Medical Science and Nutrition Salvador Zubirán. Lack of evidence did not help to establish the international recommended daily intake. Currently, vitamin D deficiency must be recognized as a health problem, worthy of attention and action. We suggest that prospective studies are carried out in our country where the relationship between serum vitamin D deficiency and poor bone mineralization will be established.

Obstrucción de la vía aérea por higroma quístico en un recién nacido / Airway obstruction due to cystic hygroma in a newborn

Introducción: El higroma quístico es una dilatación difusa de los conductos linfáticos. Puede diagnosticarse prenatalmente a través de una ecografía obstétrica. La incidencia aproximada es de 1/6,000 nacidos vivos y de 1/750 abortos espontáneos. Esta lesión puede presentarse a nivel cervical en la parte inferolateral del cuello, donde aparece con grandes cavidades únicas o multiloculares. En general, se producen por la falta de conexión de los vasos linfáticos con los sacos linfáticos yugulares, o de estos con el sistema de drenaje venoso. Caso clínico: Con el fin de enfatizar sobre la notificación de estas enfermedades y las opciones de tratamiento no quirúrgico, se presenta una paciente con higroma quístico cervical (cara lateral del cuello) con compromiso de la vía aérea y digestiva por la extensión del tumor. Se trató con etanol puro por medio de múltiples infiltraciones guiadas por ultrasonido. Conclusiones: Dependiendo de las características de la lesión, el tratamiento puede ser quirúrgico, farmacológico o mixto. Cuando la extensión es importante o se relaciona con órganos vitales, la mejor opción de tratamiento es, en primer lugar, reducir el tamaño de la lesión y el compromiso de los órganos contiguos. Esto se hace por medio de escleroterapia. Posteriormente, de ser necesario, se realiza cirugía.

Background: Cystic hygroma is a diffuse dilatation of the lymphatic system, which can be prenatally diagnosed by ultrasound. The incidence is 1/6,000 live births and 1/750 spontaneous abortions. This malformation can occur at the cervical level located in the inferior lateral part of the neck where it appears with large single or multilocular cavities. It is generally caused by a lack of connection with jugular lymphatic channels or with the venous drainage system lymph sacs. Case report: In order to emphasize these diseases and non-surgical treatment options, we present a patient with a cervical cystic hygroma that compromises the airway and digestive tract due to tumor extension and treatment with pure ethanol with clinical improvement. Conclusions: Depending on the characteristics of the lesion, treatment options are surgery, pharmacological or mixed. When the extension involves vital organs, the best option is to reduce the size of the lesion and the compromise of the adjacent organ. This is done by sclerotherapy and, if necessary, surgery.

[Airway obstruction due to cystic hygroma in a newborn]. / Obstrucción de la vía aérea por higroma quístico en un recién nacido.

BACKGROUND: Cystic hygroma is a diffuse dilatation of the lymphatic system, which can be prenatally diagnosed by ultrasound. The incidence is 1/6,000 live births and 1/750 spontaneous abortions. This malformation can occur at the cervical level located in the inferior lateral part of the neck where it appears with large single or multilocular cavities. It is generally caused by a lack of connection with jugular lymphatic channels or with the venous drainage system lymph sacs. CASE REPORT: In order to emphasize these diseases and non-surgical treatment options, we present a patient with a cervical cystic hygroma that compromises the airway and digestive tract due to tumor extension and treatment with pure ethanol with clinical improvement. CONCLUSIONS: Depending on the characteristics of the lesion, treatment options are surgery, pharmacological or mixed. When the extension involves vital organs, the best option is to reduce the size of the lesion and the compromise of the adjacent organ. This is done by sclerotherapy and, if necessary, surgery.