ABSTRACT
Objective:To investigate the genetic etiology of a Marfan syndrome pedigree, and the impact of c.4336G>A variant on the splicing process of FBN1 gene.Methods:The proband was admitted to the Department of Cardiovascular Surgery of Xijing Hospital due to thoracic aortic aneurysm and dissection in August 2019. Multiplex PCR and next generation sequencing technology were used to detect 15 genes associated with hereditary aortic diseases in the proband. Then the pathogenic sites were further verified by Sanger sequencing, and above examinations were also performed among the family members of the proband. The effect of the mutation on mRNA splicing was predicted by splicing prediction software. RNAs from peripheral blood cells of the proband and the healthy person were extracted, and the effect of the mutation on mRNA splicing was verified by reverse transcription PCR and Sanger sequencing. The pathogenicity was analyzed by the recommendations from the American College of Medical Genetics (ACMG).Results:The gene panel detected a missense mutation of FBN1 gene (c.4336G>A) in the proband. Sanger sequencing results were consistent with that of panel. Sanger sequencing results showed that 4 family members were carriers of the same variant, and 3 out of the 4 family members presented signs of thoracic aortic aneurysm and dissection. The dbscSNV_ada_score and dbscSNV_rf_score software predicted that this mutation would lead to the occurrence of abnormal splicing of mRNA. The skipping of exon 35 was verified in the subsequent examinations by reverse transcription PCR and Sanger sequencing. The variant was classified as"pathogenic"according to ACMG guideline.Conclusion:FBN1 c.4336G>A mutation can cause the skipping of exon 35, and this might be the genetic mechanistic of severe cardiovascular abnormalities observed in this Marfan syndrome pedigree.
ABSTRACT
Extracellular vesicles (EVs) can carry a variety of bioactive components including nucleic acids, proteins and small molecule metabolites, and their value in tumor diagnosis and treatment has been widely recognized. However, current studies on EV inclusions mainly focus on RNA and protein, and the role of small molecule metabolites that can most directly reflect the cell state in EV remains unclear. EV metabolomics in cancer research has gradually gained traction in recent years. There are still many challenges in EV metabolomics research due to the complexity of pretreatment and low content of metabolite, but its value in regulating tumor progression and serving as tumor markers has gradually emerged, which is expected to provide new targets for tumor diagnosis and treatment.
ABSTRACT
As a new type of intercellular signaling rector, extracellular vesicles (EV) are involved in almost the whole process of tumorigenesis, progression, metastasis, and drug resistance. Therefore, EV have become the ideal biomarker candidates and research hotspots for cancer diagnosis and treatment. However, EV tumor biomarker research mainly focused on RNA and protein, and a small part of the research focused on lipids at the early stage. EV DNA has received little attention and its diagnostic value has gradually been recognized in recent years. Study on the biological characteristics and function of EV DNA may highlight its potential in tumor diagnosis.
ABSTRACT
Extracellular vesicles (EV) are widely distributed, rich in content, and fully participate in the regulation of important cellular physiological activities. As a new technology for tumor diagnosis, the natural advantages of EV include protecting its contents, being easy to obtain from various biofluids, having the potential to be truly non-invasive and high tissue fidelity. In order to transfer EV for clinical use, many isolation technologies such as microfluidics, immunochips, nano-flow cytometry and acoustic capture have been continuously developed. At present, several EV tumor diagnostic products have been applied in the clinical setting. However, due to the great differences in detection methods and platforms, there are still some technical difficulties in its clinical applications. With the accumulation of clinical evidence and the continuous improvement of clinical diagnostic methods, the liquid biopsy based on EV can finally be applied in the clinic.
ABSTRACT
As a new type of paracrine/autocrine and remote signaling, extracellular vesicles (EVs) involved in almost the whole process of tumorigenesis, progression, metastasis, and drug resistance, which has greatly inspired tumor diagnosis and treatment. EVs have the great potential to be the blood or urine biomarkers and can be used for cancer diagnosis, prognosis and monitoring. Due to its great clinical application value, EVs have become a hotspot in cancer research in recent years. Therefore, in this review, the advantages, progression, and technical challenges of EVs biomarkers in clinical tumors diagnosis are discussed.
ABSTRACT
As a new type of paracrine/autocrine and remote signaling, extracellular vesicles (EVs) involved in almost the whole process of tumorigenesis, progression, metastasis, and drug resistance, which has greatly inspired tumor diagnosis and treatment. EVs have the great potential to be the blood or urine biomarkers and can be used for cancer diagnosis, prognosis and monitoring. Due to its great clinical application value, EVs have become a hotspot in cancer research in recent years. Therefore, in this review, the advantages, progression, and technical challenges of EVs biomarkers in clinical tumors diagnosis are discussed.
ABSTRACT
Objective@#To optimize the existing methods of isolation and purification for exosomes from urine and explore the effects of different storage conditions on the content of exosomal RNA in urine. @*Methods@#The exosomes in human urine samples were extracted by different precipitation method, i.e., precipitation following first concentrating and direct precipitation, respectively, and the separation efficiency and cost of the two methods were compared. ExoQuick-TCTM precipitation kit was used to extract exosomes. Nanoparticle tracking analysis technique (NTA) was used to detect the concentration and particle size distribution of exosome. Dynamic light scattering (DLS) was used to detect the potential of exosome. Transmission electron microscopy (TEM) was used to observe morphology of exosomes. western blot was used to analyze the exosomal marker molecules CD63 and Alix. The extraction method of the precipitation following first concentrating was used to verify the reliability of the optimized method in 10 clinical urine samples . Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of exosomal RNA marker let-7c and PSA mRNA in the urinary exosomes from 20 patients with prostate cancer after repeated freeze-thaw (0 [i.e., fresh], 1 , 3 and 5 times) and 9 patients with prostate cancer frozen at -80 ℃ for different time (0 [i.e., fresh], 1, 2 and 4 weeks), and were statistically analyzed by Wilcoxon rank sum test for differences between the 2 groups. @*Results@#The size distribution of exosomes extracted by the two methods was 30 to 150 nm by NTA, both of which were displayed as single peaks. The results of DLS showed that the potentials of exosome extracted by the two methods were negative values. The size of the exosomes extracted by the two methods was consistent observed under TEM namely the diameter distribution was 30 to 150 nm. western blot analysis confirmed that CD63 and Alix, the exosome labeling molecules, existed in the optimized method. The concentration of exosomes extracted from the 10 urine samples all reached 10 9 to 10 11 particles/mL. The contents of let-7c and PSA mRNA in exosomes decreased significantly after 5 freeze-thaw cycles, and the Z values were -1.79 and -1.73, respectively (P＜0.05). The RNA content of the exosomes remained stable after freezing at -80 ℃ for 1 month. @*Conclusion@#The optimized exosome extraction method could reduce greatly the cost under the premises of ensuring the concentration and quality of exosomes. The isolated exosomes may keep stable RNA content after freezing at -80 ℃ for a short time, but could not be frozen and thawed repeatedly for more than 5 times.
ABSTRACT
Circulating tumor DNA (ctDNA) in the blood of cancer patients provides an opportunity for non-invasive sampling of tumor DNA.This "liquid biopsy"allows for detection of copy number variations,tumor-specific mutations,epigenetic changes,and can be used to guide and improve treatment throughout real-time "tracking" the course of tumor disease.Aberrant methylation of specific gene regions can be a very consistent feature of cancer,in contrast to mutations,which makes ctDNA methylation amenable to the design of widely clinical applications in early diagnosis,monitoring disease status,predicting treatment response and predicting prognosis.Therefore,ctDNA methylation detection is considered as one of the most valuable methods for cancer diagnosis and risk assessment.