Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

Recent advances and applications of base editing systems / 生物工程学报

The CRISPR system is able to accomplish precise base editing in genomic DNA, but relies on the cellular homology-directed recombination repair pathway and is therefore extremely inefficient. Base editing is a new genome editing technique developed based on the CRISPR/Cas9 system. Two base editors (cytosine base editor and adenine base editor) were developed by fusing catalytically disabled nucleases with different necleobase deaminases. These two base editors are able to perform C>T (G>A) or A>G (T>C) transition without generating DNA double-stranded breaks. The base editing technique has been widely used in gene therapy, animal models construction, precision animal breeding and gene function analysis, providing a powerful tool for basic and applied research. This review summarized the development process, technical advantages, current applications, challenges and perspectives for base editing technique, aiming to help the readers better understand and use the base editing technique.

Does Oral Hygiene Influence Salivary pH, Lactate, and IL-1β of Basketball Players During Intense Exercise? / La Higiene Oral Influye en el pH Salival, el Lactato y la IL-1β de los Jugadores de Baloncesto Durante el Ejercicio Intenso?

ABSTRACT: This study examined the salivary pH, salivary lactate, and salivary IL-1 β responses from a high-intensity intermittent running test, and the influence of hygiene oral status on these biomarkers in elite adolescent basketball players. Forty-six adolescent players participated. Saliva sampling was taken before and 3 min after a high-intensity exercise (Yo-Yo Intermittent Recovery Test Level 1; Yo-Yo IR1). In order to quantify and classify the oral hygiene level, the athletes were submitted to a dental examination, and an adapted Simplified Oral Hygiene Index was applied. After the dental examination, the whole group was divided into good oral hygiene group (GHG) and poor oral hygiene group (PHG). The results of a two- way analysis of variance showed a significant interaction effect (P = 0.0003), group effect (P < 0.0001), and time effect (pre to post Yo-yo IR1; P < 0.0001) for salivary pH and for salivary lactate (interaction effect, P = 0.008; group effect, P < 0.000 1; time effect, P < 0.0001) with a lower salivary pH and a higher salivary lactate at pre and post-Yo-Yo IR1 for PHG, but no difference was observed for IL-1β. The data demonstrated that the high-intensity exercise led to a significant change in salivary pH and salivary lactate concentration of the basketball players, and that the oral hygiene status influenced these responses, with a greater change for those players showing a poor oral hygiene.

RESUMEN: Este estudio examinó las respuestas de pH salival, lactato salival e IL-1β salival de una prueba de carrera intermitente de alta intensidad, y la influencia del estado de higiene oral en los biomarcadores en jugadores adolescentes de baloncesto de élite. En el análisis participaron 46 jugadores adolescentes. Se tomó una muestra de saliva antes y 3 minutos después de un ejercicio de alta intensidad (Prueba de recuperación intermitente Yo-Yo Nivel 1; Yo-Yo IR1). Para cuantificar y clasificar el nivel de higiene oral, los atletas fueron sometidos a un examen dental y se aplicó un índice adaptado de higiene oral simplificado. Después del examen dental, el grupo se dividió en un grupo de buena higiene oral (GHG) y un grupo de mala higiene oral (PHG). Los resultados de un análisis de varianza mostraron un efecto de interacción significativo (P = 0.0003), efecto de grupo (P<0.0001) y efecto de tiempo (antes y después de Yo-yo IR1; P <0.0001) para el pH salival y para lactato salival (efecto de interacción, P = 0.008; efecto de grupo, P <0.0001; efecto de tiempo, P <0.0001) con pH salival más bajo y lactato salival más alto en IR1 pre y post YoY para PHG, pero no se observó una diferencia para IL-1β. Los datos demostraron que el ejercicio de alta intensidad genera un cambio significativo en el pH salival y el lactato de los jugadores de baloncesto, y que el estado de higiene oral influyó en estas respuestas, con un cambio mayor para aquellos jugadores que mostraron una mala higiene oral.

Avaliação dos níveis solúveis de sTIM-3, s4-1BB e de citocinas inflamatórias em mulheres com câncer de mama triplo negativo localmente avançado submetidas a quimioterapia neoadjuvante / Evaluation of soluble levels of sTIM-3, s4-1BB and inflammatory cytokines in women with locally advanced triple negative breast cancer submitted to neoadjuvant chemotherapy

Introdução: O câncer de mama triplo negativo (CMTN) corresponde de 10 a 20% dos carcinomas invasivos de mama, é altamente agressivo e na maioria das vezes tem apenas a quimioterapia padrão como tratamento. As citocinas inflamatórias são um grupo heterogêneo de proteínas solúveis produzidas por diferentes tipos de células, que medeiam e regulam o sistema imune. A resposta imune celular é regulada por proteínas acessórias denominadas receptores co-estimuladores e co-inibidores, como 4-1BB e TIM-3, respectivamente. A procura por biomarcadores que possam predizer resposta à quimioterapia neoadjuvante (QT neo) ainda é um desafio na medicina. Objetivo: Avaliar os níveis solúveis de sTIM-3 e s4-1BB, e de citocinas inflamatórias no sangue de mulheres com câncer de mama triplo negativo localmente avançado e associá-los à sobrevida livre de doença e com o tipo de resposta patológica à QT neo. Métodos: O estudo foi realizado entre os anos de 2015 e 2017 no Hospital de Câncer de Pernambuco (HCP) e Laboratório de Pesquisa Translacional do Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Foram incluídas 29 mulheres, entre 18 e 60 anos de idade, com diagnóstico de CMTN localmente avançado e submetidas à QT neo, e um grupo de 30 mulheres saudáveis. Coletas de sangue periférico foram realizadas antes e após a QT neo. A dosagem dos níveis solúveis de s4-1BB e sTIM-3 foi realizada por enzyme linked immunonoSorbent assay (ELISA). As dosagens de IL-1ß, IL-6, IL-8, TNF-α e IL-10 foram realizadas pela técnica de Cytometric Bead Array por citometria de fluxo. O nível de significância estatística foi de p<0,05. Resultados: Níveis mais elevados de IL-6 e de IL-10 foram observados no grupo de pacientes com tumor T4 com relação as grupo T2-T3 (p<0.05). Níveis elevados de IL-6, IL-8, IL-10 e TNF-α no grupo CMTN com status linfonodal N2 versus N0 e N1 (p<0,05). Os níveis de IL-1ß, IL-6, IL-10 e TNF-α foram elevados no grupo com resposta patológica parcial (RP) quando comparado aos grupos com resposta patológica completa (RC) e controles (p<0,05). Com relação aos níveis de IL-8, os grupos de pacientes RC e RP apresentaram níveis elevados quando comparados aos controles (p<0,05). Não foi observada diferença significativa de IL-8 entre os grupos RC e RP. Não foram observadas diferenças significativas em níveis de s4-1BB E sTIM-3 de acordo com o tumor primário e status linfonodal. Elevados níveis de s4-1BB foram observados no grupo RC comparado aos grupos RP e de controles (p<0,0004 e p<0,0001, respectivamente). Da mesma forma, os níveis de sTIM-3 foram mais elevados nas pacientes com RC e RP em relação aos controles (p<0,0001 e p<0,0003, respectivamente). Para análise da sobrevida livre de doença (SLD) em 24 meses de seguimento, utilizamos como ponto de corte o valor da mediana (< ou ≥ percentil 50) dos níveis solúveis de sTIM-3 e s4-1BB. Não houve diferenças significativas na SLD entre os grupos com níveis de s4-1BB ≥ 122 e < 122 pg/mL. A SLD foi de 93,33% no grupo com níveis de sTIM-3 < 2874 pg/mL e de 60% no grupo com níveis ≥ 2874 pg/mL (p=0,03). Conclusão: O s4-1BB foi um bom indicador preditivo de resposta à QT neo, enquanto elevação de sTIM-3 mostrou estar associado ao risco de recidiva loco-regional e metástases à distância. Ambos, TIM-3 e 4-1BB, parecem ser potenciais alvos terapêuticos no CMTN localmente avançado por estarem associadas ao desfecho clínico da doença

Introduction: Triple negative breast cancer (TNBC) corresponds to 10 to 20% of invasive breast carcinoma with high aggressiveness and in most cases has only standard chemotherapy as treatment. Inflammatory cytokines are heterogeneous group of soluble proteins produced by different types of cell, that mediate and regulate the immune system. The cellular immune response is regulated by accessory proteins called co-stimulatory and co-inhibitory receptors, such as 4-1BB and TIM-3, respectively. The search for biomarkers that can predict response to neoadjuvant chemotherapy (NAC) is still a challenge to medicine. Objective: To evaluate the soluble levels of sTIM-3 and s4-1BB, and inflammatory cytokines in the blood of women with locally advanced triple negative breast cancer, and to associate them with disease-free survival and the type of pathological response to NAC. Methods: The study was carried out between the years 2015 and 2017 at the Hospital de Cancer de Pernanbuco (HCP) and Translational Research Laboratory of the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Twenty-nine women, between 18 and 60 years old, diagnosed with locally advanced TNBC, and submitted to NAC, and 30 healthy women were included. Peripheral blood samples were taken before and after neo QT. The determination of the soluble levels of s4-1BB and sTIM-3 was performed by enzyme linked immunonosorbent assay (ELISA). The measurements of IL-1ß, IL-6, IL-8, TNF-α and IL-10 were performed using the Cytometric Bead Array by flow cytometry. The level of statistical significance was p <0.05. Results: There were higher levels of IL-6 and IL-10 in the group of patients with tumor size T4 compared to groups T2-T3 (p <0.05). High levels of IL-6, IL8, IL-10 and TNF-α were found in the TNBC group with lymph node status in N2 in relation to N0 and N1 (p <0.05). High levels of IL1ß, IL-6, IL-10 and TNF-α were observed in the group with partial pathological response (PR) when compared to groups with complete pathologial response (RC) and controls (p <0.05). With regard to IL-8 levels, the groups of CR and RP patients showed high levels when compared to controls (p <0.05). There was no significant difference in IL-8 between the CR and PR groups. No significant differences were observed in the levels of s4-1BB and sTIM-3 in accordance with primary tumor and lymph node status. High levels of s4-1BB were observed in the RC group compared to the PR and control groups (p <0.0004 and p <0.0001, respectively). As well, the levels of sTIM-3 were higher in patients with CR and PR compared to controls (p <0.0001 and p <0.0003, respectively). In the analyses of disease-free survival (DFS) with 24 months of follow-up, we used as a cut-off point the median value (< or ≥ 50th percentile) of the soluble levels of sTIM-3 and s4-1BB. There were no significant differences in DFS at 24 months between groups with levels of s4-1BB ≥ 122 and <122 pg/mL. The DFS was 93.33% in the group with sTIM-3 levels <2874 pg/mL and 60% in the group with levels ≥ 2874 pg/mL (p = 0.03). Conclusion: s4-1BB was a good predictive indicator of response to NAC, while elevation of sTIM-3 levels was shown to be associated with the risk of locoregional recurrence and distant metastases. Both TIM-3 and 4-1BB appear to be potential therapeutic targets in locally advanced TNBC because they were associated with the clinical outcome of the disease

Comparison of the phenolic contents and epigenetic and genetic variability of wild and cultivated watercress (Rorippa nasturtium var. aquaticum L.)

Background: Epigenetic modifications are key factors modulating the expression of genes involved in the synthesis of phytochemicals. The knowledge of plant epigenetic and genetic variations can contribute to enhance the production of bioactive compounds. These issues have been little explored thus far in Rorippa nasturtium var. aquaticum L. (watercress), an edible and medicinal plant. The aim of the current study was to determine and compare the phenolic composition and epigenetic and genetic variations between wild and cultivated watercress. Results: Significant differences were found in the quantitative phenolic composition between wild and cultivated watercress. The eight primer combinations used in the methylation-sensitive amplification polymorphism (MSAP) method revealed different epigenetic status for each watercress type, the cultivated one being the most epigenetically variable. The genetic variability revealed by the EcoRI/MspI amplification profile and also by eight inter-simple sequence repeat (ISSR) primers was different between the two types of watercress. The results of the Mantel test showed that the correlation between genetic and epigenetic variations has diminished in the cultivated type. Cluster analyses showed that the epigenetic and genetic characterizations clearly discriminated between wild and cultivated watercress. Conclusions: Relevant chemical, epigenetic, and genetic differences have emerged between wild and cultivated watercress. These differences can contribute to fingerprint and develop quality control tools for the integral and safety use and the commercialization of watercress. The richness of epialleles could support the development of tools to manipulate the watercress epigenome to develop high bioproduct­producing cultivars

El caseinato de sodio incrementa número de linfocitos B en ratones / Sodium caseinate increases the number of B lymphocytes in mouse

Introducción. El caseinato de sodio, una sal de la caseína utilizada como agente proinflamatorio en ratones, es capaz de inducir granulopoyesis en vivo e incrementar la producción de citocinas esenciales en dicho evento. Objetivo. Evaluar si el caseinato de sodio es capaz de inducir un efecto biológico en células de origen linfoide y la producción de citocinas involucradas con este linaje. Materiales y métodos: Se utilizaron ratones hembra BALB/c de 8 a 12 semanas de edad. Los animales se inyectaron cuatro veces, con intervalos de 48 horas, por vía intraperitoneal con 1 ml de caseinato de sodio (10 % de SFB p/v). La población de linfocitos B y la incorporación de bromodesoxiuridina (BrdU) se analizaron mediante citometría de flujo. La detección de la interleucina 7 se evaluó mediante la técnica de ELISA. Resultados. Tras la inyección por vía intraperitoneal, el número de linfocitos B 220+ provenientes del bazo de ratones tratados con caseinato de sodio aumentó comparados con los que solo recibieron el vehículo como tratamiento (89,01±1,03 Vs. 75,66±2,08), así como la incorporación de BrdU en células B220+ (38,59±4,48 Vs. 11,82±1,04). Se evidenció, asimismo, el incremento en la concentración de la interleucina 7 (IL-7) en el suero de los ratones tratados con caseinato de sodio, comparados con los que solo recibieron el vehículo (62,1±17,5 Vs. 26,9±4,4 pg/ml). Conclusión. El caseinato de sodio fue capaz de aumentar el número de linfocitos B en bazo de ratones, así como inducir la producción de IL-7, citocina clave para la linfopoyesis B.

Introduction: Sodium caseinate, a casein salt, is a proinflammatory agent in mice, and it is able to induce granulopoiesis in vivo and to increase the production of cytokines, which is key for this biological process. Objective: To assess whether sodium caseinate is able to induce a biological effect on cells from lymphoid origin and the production of cytokines involved in this lineage in vivo. Materials and methods: We used female BALB /c mice from 8 to 12 weeks old. The animals were injected intraperitoneally (IP) with 1 ml of sodium caseinate (10% PBS w/v) four times every 48 hours. The B cell populations and the incorporation of BrdU were analyzed by flow cytometry. Detection of interleukin-7 was assessed by ELISA (Enzyme-Linked ImmunoSorbent Assay). Results: We established that after intraperitoneal injection, the number of B lymphocytes 220+ from the spleen of mice treated with sodium caseinate increased compared to those that only received the vehicle (89.01±1.03 vs 75.66 ± 2.08), and the same was observed with the incorporation of BrdU in B220 + cells (38.59±4.48 vs 11.82±1.04 respectively). We also established that the concentration of interleukin-7 (IL-7) in the serum of mice treated with sodium caseinate increased compared to those that only received the vehicle (62.1 ± 17.5 vs 26.9 ± 4.4 pg/ml). Conclusion: Sodium caseinate was able to increase the number of B lymphocytes in the spleen; it also induced IL-7 production, a cytokine that is key for the B cell lymphopoiesis.

TET family dioxygenases and DNA demethylation in stem cells and cancers

The methylation of cytosine and subsequent oxidation constitutes a fundamental epigenetic modification in mammalian genomes, and its abnormalities are intimately coupled to various pathogenic processes including cancer development. Enzymes of the Ten-eleven translocation (TET) family catalyze the stepwise oxidation of 5-methylcytosine in DNA to 5-hydroxymethylcytosine and further oxidation products. These oxidized 5-methylcytosine derivatives represent intermediates in the reversal of cytosine methylation, and also serve as stable epigenetic modifications that exert distinctive regulatory roles. It is becoming increasingly obvious that TET proteins and their catalytic products are key regulators of embryonic development, stem cell functions and lineage specification. Over the past several years, the function of TET proteins as a barrier between normal and malignant states has been extensively investigated. Dysregulation of TET protein expression or function is commonly observed in a wide range of cancers. Notably, TET loss-of-function is causally related to the onset and progression of hematologic malignancy in vivo. In this review, we focus on recent advances in the mechanistic understanding of DNA methylation-demethylation dynamics, and their potential regulatory functions in cellular differentiation and oncogenic transformation.

Anti-proliferative Effect of Engineered Neural Stem Cells Expressing Cytosine Deaminase and Interferon-β against Lymph Node–Derived Metastatic Colorectal Adenocarcinoma in Cellular and Xenograft Mouse Models / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Genetically engineered stem cells may be advantageous for gene therapy against various human cancers due to their inherent tumor-tropic properties. In this study, genetically engineered human neural stem cells (HB1.F3) expressing Escherichia coli cytosine deaminase (CD) (HB1.F3.CD) and human interferon-β (IFN-β) (HB1.F3.CD.IFN-β) were employed against lymph node–derived metastatic colorectal adenocarcinoma. MATERIALS AND METHODS: CD can convert a prodrug, 5-fluorocytosine (5-FC), to active 5-fluorouracil, which inhibits tumor growth through the inhibition of DNA synthesis,while IFN-β also strongly inhibits tumor growth by inducing the apoptotic process. In reverse transcription polymerase chain reaction analysis, we confirmed that HB1.F3.CD cells expressed the CD gene and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β genes. RESULTS: In results of a modified trans-well migration assay, HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward SW-620, human lymph node–derived metastatic colorectal adenocarcinoma cells. The viability of SW-620 cells was significantly reduced when co-cultured with HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. In addition, it was found that the tumor-tropic properties of these engineered human neural stem cells (hNSCs) were attributed to chemoattractant molecules including stromal cell-derived factor 1, c-Kit, urokinase receptor, urokinase-type plasminogen activator, and C-C chemokine receptor type 2 secreted by SW-620 cells. In a xenograft mouse model, treatment with hNSC resulted in significantly inhibited growth of the tumor mass without virulent effects on the animals. CONCLUSION: The current results indicate that engineered hNSCs and a prodrug treatment inhibited the growth of SW-620 cells. Therefore, hNSC therapy may be a clinically effective tool for the treatment of lymph node metastatic colorectal cancer.

Improvement and Evaluation on Bisulfite Modification of DNA for DNA Methylation Detection / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a rapid and convenient method of DNA modification by bisulfite sodium for the detection of DNA methylation.</p><p><b>METHODS</b>Through increasing the bisulfite sodium concentration and the temperature of treatment, cutting down the modification time, besides using glassmilk to adsorb the DNA in the purification and recovery, to improve the methods of DNA modification. Efficiency of cytosine converted to thymine in MAGE-A3 gene and DAP-K gene fragments were analyzed by bisulfite sequencing PCR in order to evaluate the DNA modification effect among the improved method, traditional method and kit method.</p><p><b>RESULTS</b>The operating time of test was shortened to about 3 hours by the improved method; conversion rate of unmethylated cytosine to thymine was over 99%; compared with the traditional method and kit method, there was no significant difference (χ(2) = 0.0564, P > 0.05); the improved method was only for the unmethylated cytosine conversion modification, and there was no significant difference in process of methylated cytosine converted to thymine comparing with the traditional method (χ(2) = 0.0149, P > 0.05).</p><p><b>CONCLUSION</b>The improved method has high efficiency of DNA modification and has no significant effect on excessive modification;meanwhile, it has many advantages such as time-saving and easy to operate etc.</p>

Possible Role of a Missense Mutation of p.P167S on NOTCH3 Gene Associated with Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a single-gene disorder caused by mutations in the NOTCH3 gene, located on chromosome 19p13. NOTCH3 encodes a transmembrane receptor which plays a role in cellular differentiation and cell cycle regulation. CASE REPORT: A 71-year-old female showing headache and memory impairment, familial history of stroke and having a missense mutation from proline to serine at codon 167 in the exon 4 on NOTCH3 gene. Five family members revealed the same mutation (c.499C>T), who presented migrainous headache and stroke. In this study, we have uncovered a novel NOTCH3 mutation at the nucleotide position 499 (c.499C>T; p.P167S) in a family with CADASIL. CONCLUSIONS: We suggested a missense mutation of proline to serine at codon 167 in exon 4 of the NOTCH3 gene, which resulted in the substitution of cytosine to thymine (c.499C>T) resulting migraine, stroke and vascular cognitive impairment.

A Pedigree with c.179 Cytosine to Threonine Missense Mutation of SLC12A3 Gene Presenting Gitelman's Syndrome

A 42-year-old man came to the hospital presenting chest discomfort and general weakness. He had come to the hospital with the same symptoms 3 months ago and 12 years prior. His laboratory test showed hypokalemia, hypomagnesemia and hypocalciuria. The arterial blood gas analysis showed hypochloremic metabolic alkalosis. He had an ultrasonography guided renal biopsy, the result was normal at light microscopy and immunofluorescence microscopy. However, a special stain for Na-Cl cotransporter was weakly expressed compared with the control. The patient and his family underwent genetic sequencing about the SLC12A3 gene. He had a homozygous mutation in the 179th nucleotide of Exon 1 on the SLC12A3 gene (p.Thr60Met) and his parents and sisters were diagnosed as carrier state of Gitelman's syndrome (GS). GS is an inherited tubular disorder which presents mild hypokalemia, hypomagnesemia and hypocalciuria. Since the symptoms and laboratory results are not severe, it can go unnoticed by physicians. Herein we present a family with GS, diagnosed by genetic sequencing.

A novel FBN1 gene mutation associated with early-onset pneumothorax in Marfan syndrome

Marfan syndrome (MFS) is an inherited connective tissue disorder with a mutation in the fibrillin-1 (FBN1) gene. Fibrillin is a major building block of microfibrils, which constitute the structural component of the connective tissues. A 10-year-old girl visited our hospital with the chief complaint of precocious puberty. According to her medical history, she had a pulmonary wedge resection for a pneumothorax at 9 years of age. There was no family history of MFS. Mid parental height was 161.5 cm. The patient's height was 162 cm (>97th percentile), and her weight was 40 kg (75th-90th percentile). At the time of initial presentation, her bone age was approximately 11 years. From the ophthalmologic examination, there were no abnormal findings except myopia. There was no wrist sign. At the age of 14 years, she revisited the hospital with the chief complaint of scoliosis. Her height and weight were 170 cm and 50 kg, respectively, and she had arachnodactyly and wrist sign. We performed an echocardiograph and a test for the FBN1 gene mutation with direct sequencing of 65 coding exons, suspecting MFS. There were no cardiac abnormalities including mitral valve prolapse. A cytosine residue deletion in exon 7 (c.660delC) was detected. This is a novel mutation causing a frameshift in protein synthesis and predicted to create a premature stop codon. We report the case of a patient with MFS with a novel FBN1 gene missense mutation and a history of pneumothorax at a young age without cardiac abnormalities during her teenage years.

Iron Supplementation Reverses the Reduction of Hydroxymethylcytosine in Hepatic DNA Associated With Chronic Alcohol Consumption in Rats

BACKGROUND: Alcohol is known to affect two epigenetic phenomena, DNA methylation and DNA hydroxymethylation, and iron is a cofactor of ten-eleven translocation (TET) enzymes that catalyze the conversion from methylcytosine to hydroxymethylcytosine. In the present study we aimed to determine the effects of alcohol on DNA hydroxymethylation and further effects of iron on alcohol associated epigenetic changes. METHODS: Twenty-four male Sprague-Dawley rats were fed either Lieber-DeCarli alcohol diet (36% calories from ethanol) or Lieber-DeCarli control diet along with or without iron supplementation (0.6% carbonyl iron) for 8 weeks. Hepatic non-heme iron concentrations were measured by colorimetric assays. Protein levels of hepatic ferritin and transferrin receptor were determined by Western blotting. Methylcytosine, hydroxymethylcytosine and unmodified cytosine in DNA were simultaneously measured by liquid chromatography/mass spectrometry method. RESULTS: Iron supplementation significantly increased hepatic non-heme iron contents (P < 0.05) but alcohol alone did not. However, both alcohol and iron significantly increased hepatic ferritin levels and decreased hepatic transferrin receptor levels (P < 0.05). Alcohol reduced hepatic DNA hydroxymethylation (0.21% ± 0.04% vs. 0.33% ± 0.04%, P = 0.01) compared to control, while iron supplementation to alcohol diet did not change DNA hydroxymethylation. There was no significant difference in methylcytosine levels, while unmodified cytosine levels were significantly increased in alcohol-fed groups compared to control (95.61% ± 0.08% vs. 95.26% ± 0.12%, P = 0.03), suggesting that alcohol further increases the conversion from hydroxymethylcytosine to unmodified cytosine. CONCLUSIONS: Chronic alcohol consumption alters global DNA hydroxymethylation in the liver but iron supplementation reverses the epigenetic effect of alcohol.

Rabdomiólisis grave secundaria a deshidratación hipernatrémica / Severe rhabdomyolysis secondary to severe hypernatraemic dehydration

Introducción: La rabdomiólisis es una enfermedad poco frecuente en pediatría. El objetivo es presentar un paciente en el que se desarrolló secundario a una deshidratación hipernatrémica grave tras una diarrea aguda. Caso clínico: Lactante de 11 meses que consultó por fiebre, vómitos, diarrea y anuria. Presentó convulsión tónico-clónica autolimitada. Ingresó en mal estado general, severamente deshidratado, con escasa reactividad. En las pruebas complementarias destacó acidosis metabólica grave, hipernatremia e insuficiencia renal prerrenal. Al tercer día apreció leve hipotonía axial y elevación de creatín fosfokinasa 75.076 UI/l, interpretado como rabdomiólisis. Se inició hiperhidratación y alcalinización sistémica, con buena respuesta clínica y bioquímica, siendo dado de alta sin secuelas motoras. Conclusiones: La hipernatremia grave está descrita como causa rara de rabdomiólisis e insuficiencia renal. En pacientes críticos es importante un alto índice de sospecha de rabdomiólisis y determinación seriada de la creatín fosfokinasa para su detección y tratamiento precoz.

Introduction: Rhabdomyolysis is a rare paediatric condition. The case is presented of a patient in whom this developed secondary to severe hypernatraemic dehydration following acute diarrhoea. Case report: Infant 11 months of age who presented with vomiting, fever, diarrhoea and anuria for 15 hours. Parents reported adequate preparation of artificial formula and oral rehydration solution. He was admitted with malaise, severe dehydration signs and symptoms, cyanosis, and low reactivity. The laboratory tests highlighted severe metabolic acidosis, hypernatraemia and pre-renal kidney failure (Sodium [Na] plasma 181 mEq/L, urine density> 1030). He was managed in Intensive Care Unit with gradual clinical and renal function improvement. On the third day, slight axial hypotonia and elevated cell lysis enzymes (creatine phosphokinase 75,076 IU/L) were observed, interpreted as rhabdomyolysis. He was treated with intravenous rehydration up to 1.5 times the basal requirements, and he showed a good clinical and biochemical response, being discharged 12 days after admission without motor sequelae. Conclusions: Severe hypernatraemia is described as a rare cause of rhabdomyolysis and renal failure. In critically ill patients, it is important to have a high index of suspicion for rhabdomyolysis and performing serial determinations of creatine phosphokinase for early detection and treatment.

Treatment of BK virus-associated hemorrhagic cystitis with low-dose intravenous cidofovir in patients undergoing allogeneic hematopoietic cell transplantation

BACKGROUND/AIMS: BK virus (BKV) has been associated with late-onset hemorrhagic cystitis (HC) in recipients of hematopoietic stem cell transplantation (HSCT). Cidofovir has been used at higher doses (3 to 5 mg/kg/wk) with probenecid prophylaxis; however, cidofovir may result in nephrotoxicity or cytopenia at high doses. METHODS: Allogeneic HSCT recipients with BKV-associated HC are treated with 1 mg/kg intravenous cidofovir weekly at our institution. A microbiological response was defined as at least a one log reduction in urinary BKV viral load, and a clinical response was defined as improvement in symptoms and stability or reduction in cystitis grade. RESULTS: Eight patients received a median of 4 weekly (range, 2 to 11) doses of cidofovir. HC occurred a median 69 days (range, 16 to 311) after allogeneic HSCT. A clinical response was detected in 7/8 patients (86%), and 4/5 (80%) had a measurable microbiological response. One patient died of uncontrolled graft-versus-host disease; therefore, we could not measure the clinical response to HC treatment. One microbiological non-responder had a stable BKV viral load with clinical improvement. Only three patients showed transient grade 2 serum creatinine toxicities, which resolved after completion of concomitant calcineurin inhibitor treatment. CONCLUSIONS: Weekly intravenous low-dose cidofovir without probenecid appears to be a safe and effective treatment option for patients with BKV-associated HC.

Analysis of C43G mutation in the promoter region of the XIST gene in patients with idiopathic primary ovarian insufficiency / 대한생식의학회지

OBJECTIVE: The XIST gene is considered to be an attractive candidate gene for skewed X-chromosome inactivation and a possible cause of primary ovarian insufficiency (POI). The purpose of this study was to investigate whether the XIST gene promoter mutation is associated with idiopathic POI in a sample of the Korean population. METHODS: Subjects consisted of 102 idiopathic POI patients and 113 healthy controls with normal menstrual cycles. Patients with the following known causes of POI were excluded in advance: cytogenetic abnormalities, prior chemo- or radiotherapy, or prior bilateral oophorectomy. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The mean age of onset of ovarian insufficiency was 28.7+/-8.5 years and the mean values of serum luteinizing and follicle-stimulating hormones and estradiol in the POI group were 31.4+/-18.2 mIU/mL, 74.5+/-41.1 mIU/mL, and 30.5+/-36.7 pg/mL, respectively. We found no cytosine to guanine (C43G) variation in the XIST gene in both POI patients and controls. CONCLUSION: The C43G mutation in the promoter region of the XIST gene was not present in the Korean patients with idiopathic POI in our study, in contrast to our expectation, suggesting that the role of XIST in the pathogenesis of POI is not yet clear.

Insulin-like growth factor 1 gene (CA)n repeats and a variable number of tandem repeats of the insulin gene in Brazilian children born small for gestational age

OBJECTIVE: To investigate the influence of (CA)n repeats in the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene on birth size in children who are small or adequate-sized for gestational age and to correlate these polymorphisms with serum insulin-like growth factor 1 levels and insulin sensitivity in children who are small for gestational age, with and without catch-up growth. PATIENTS AND METHODS: We evaluated 439 infants: 297 that were adequate-sized for gestational age and 142 that were small for gestational age (66 with and 76 without catch-up). The number of (CA)n repeat in the insulin-like growth factor 1 gene and a variable number of tandem repeats in the insulin gene were analyzed using GENESCAN software and polymerase chain reaction followed by enzymatic digestion, respectively. Clinical and laboratory data were obtained from all patients. RESULTS: The height, body mass index, paternal height, target height and insulin-like growth factor 1 serum levels were higher in children who were small for gestational age with catch-up. There was no difference in the allelic and genotypic distributions of both polymorphisms between the adequate-sized and small infants or among small infants with and without catch-up. Similarly, the polymorphisms were not associated with clinical or laboratory variables. CONCLUSION: Polymorphisms of the (CA)n repeats of the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene, separately or in combination, did not influence pre- or postnatal growth, insulin-like growth factor 1 serum levels or insulin resistance. .

Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA.

CONTEXT: Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters. AIM: Optimize a cost effective PCR assay to amplify the GC-rich DNA templates. SETTINGS AND DESIGN: Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5’ untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells. MATERIALS AND METHODS: A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase. RESULTS: Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used. CONCLUSIONS: It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases.

Participação de fatores de crescimento e de citosinas nas alterações observadas na prole de ratas tratadas com o veneno do escorpião Tityus Bahiensis no período gestacional / Participation of growth factors and citokynes in alterations observed in offspring of rats treated with Tityus bahiensis scorpion venom during the pregnancy

No Brasil, o escorpionismo é um problema de saúde pública. O escorpião T. serrulatus é considerado o mais perigoso, mas um grande número de acidentes também acontece com o T. bahiensis. O objetivo do estudo foi verificar possíveis efeitos do veneno do escorpião T. bahiensis no desempenho reprodutivo materno e nos níveis de citocinas e fatores de crescimento em embriões de mães tratadas durante a gestação. Para os parâmetros reprodutivos foram utilizadas fêmeas prenhes injetadas com uma dose de 2,5mg/Kg (s.c.) do veneno no 5º (GD5) ou no 10º (GD10) dia gestacional. O grupo controle foi injetado com salina a 1,46% (1ml/Kg) em ambos os dias. No 21º dia gestacional, os filhotes foram retirados por laparotomia e divididos em dois grupos que receberam tratamento específico para a análise visceral e esquelética. Para avaliação dos níveis de citocinas e de fatores de crescimento, as fêmeas prenhes foram injetadas com salina (1ml/Kg), LPS (100μg/kg) ou veneno (2,5mg/Kg) no 10º (GD10) ou 16º (GD16) dia gestacional. Os filhotes foram removidos por laparotomia 6, 12 ou 24 horas após o tratamento materno. As amostras foram maceradas em um homogenizador de tecido e centrifugadas. Os níveis de citocinas e fatores de crescimento foram determinados por ensaios imunoenzimáticos. Nos parâmetros reprodutivos não houve alterações no peso materno durante a gestação, no número de corpos lúteos, número de filhotes, peso do útero e dos filhotes. Houve diminuição no número de implantações e reabsorções no grupo GD5. Houve aumento no peso das placentas em GD5 e GD10. No desenvolvimento dos filhotes foram observadas aumento no peso do coração e pulmão em GD5 e GD10 e no peso do fígado em GD10. Não foram observadas anomalias e malformações internas ou externas nos filhotes de ambos os grupos experimentais. Em GD10 não foram observadas alterações nos níveis de citocinas 6 horas após a aplicação do veneno, mas verificou-se diminuição do nível de INF-γ 24 horas depois...

In Brazil scorpionism is a public health problem. The scorpion T. serrulatus is considered the most dangerous, but a large number of accidents also occur with T. bahiensis. The objective of this work was to verify the possible effects of the T. bahiensis scorpion venom on the maternal reproductive parameters and on the cytokines levels and growth factors in embryos after the treatment of pregnancy females. To the reproductive parameters it was used pregnant females injected with a dose of 2.5mg/Kg (s.c.) of the venom. The experimental groups were injected with venom on the 5th (GD5) or on the 10th (GD10) gestational day. The control group was injected with NaCl 1.46% on both days. On the 21st gestational day, the pups were taken out by laparotomy and were divided into two groups that received specific treatments for skeletal or visceral analyses. To evaluate the cytokines levels, pregnant females were injected with saline (1ml/kg), LPS (100μg/kg) or crude venom (2.5mg/kg) on the 10th (GD10) or 16th (GD16) gestational day. The pups were removed by laparotomy 6, 12 or 24 hours after the mother’s treatment. The samples (embryo/placenta) were macerated by a tissue homogenizer and centrifuged. The cytokine levels were determined by enzyme immunoassays. In the reproductive parameters no changes were observed in the maternal weight during the gestational period, corpora lutea, number of pups, uterus weight and pups weight. There was a decrease on the number of implantation and resorption in GD5 There was alteration on the placenta’s weight in GD5 and GD10. In pups development there were observed alterations in the heart and lung weight on GD5 and GD10 and on the liver weight on GD10. There were not observed external or internal anomalies and malformations in the offspring of both experimental groups. The cytokines levels were not alterated after 6 hours in GD10. In GD16 there was an increase in the IL-1α levels...