scholarly journals Functional validation of genetic variants identified by next generation sequencing in malformations of cortical development

2021 ◽  
Author(s):  
Dalila De Vita
Keyword(s):  
Next Generation Sequencing ◽  
Genetic Variants ◽  
Cortical Development ◽  
Next Generation ◽  
Malformations Of Cortical Development ◽  
Functional Studies ◽  
Generation Sequencing ◽  
In Vitro Treatment

Malformations of cortical development (MCDs) result from a disruption in the process of the human brain cortex formation: currently, there are no pharmacological treatments for diffuse MCDs. Next-generation sequencing has accelerated the identification of MCDs causing genes: in some cases, functional studies are needed to clarify the role of genetic variants. The aim of this PhD project has been to apply a multidisciplinary approach to identify causative mutations in patients with MCDs, validate the pathogenic role of the identified mutations, and assess the effectiveness of novel in vitro treatment for mTOR pathway related MCDs.

scholarly journals LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/β-catenin pathway

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Weiyuan Li ◽  
Ganggang Yang ◽  
Dengke Yang ◽  
Dong Li ◽  
Qian Sun
Keyword(s):  
Next Generation Sequencing ◽  
Rna Binding ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Normal Tissues ◽  
Factor C ◽  
Generation Sequencing

Abstract Background Long noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown. Methods Next-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1. Results The next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44. Conclusion In summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

scholarly journals LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/β-catenin pathway

2020 ◽  
Author(s):  
Weiyuan Li ◽  
Ganggang Yang ◽  
Dengke Yang ◽  
Dong Li ◽  
Qian Sun
Keyword(s):  
Next Generation Sequencing ◽  
Rna Binding ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Normal Tissues ◽  
Factor C ◽  
Generation Sequencing

Abstract Background: Long noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown.Methods: Next-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1.Results: The next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44.Conclusion: In summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

scholarly journals Environmental Surveillance through Next-Generation Sequencing to Unveil the Diversity of Human Enteroviruses beyond the Reported Clinical Cases

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 120
Author(s):  
Andrés Lizasoain ◽  
Daiana Mir ◽  
Gisella Masachessi ◽  
Adrián Farías ◽  
Nélida Rodríguez-Osorio ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Echovirus 30 ◽  
Coxsackievirus B4 ◽  
Important Region ◽  
Enterovirus A71 ◽  
Wastewater Samples ◽  
Valuable Role ◽  
Generation Sequencing

The knowledge about circulation of Human Enteroviruses (EVs) obtained through medical diagnosis in Argentina is scarce. Wastewater samples monthly collected in Córdoba, Argentina during 2011–2012, and then in 2017–2018 were retrospectively studied to assess the diversity of EVs in the community. Partial VP1 gene was amplified by PCR from wastewater concentrates, and amplicons were subject of next-generation sequencing and genetic analyses. There were 41 EVs detected, from which ~50% had not been previously reported in Argentina. Most of the characterized EVs (60%) were detected at both sampling periods, with similar values of intratype nucleotide diversity. Exceptions were enterovirus A71, coxsackievirus B4, echovirus 14, and echovirus 30, which diversified in 2017–2018. There was a predominance of types from EV-C in 2017–2018, evidencing a common circulation of these types throughout the year in the community. Interestingly, high genetic similarity was evidenced among environmental strains of echovirus 30 circulating in 2011–2012 and co-temporal isolates obtained from patients suffering aseptic meningitis in different locations of Argentina. This study provides an updated insight about EVs circulating in an important region of South America, and suggests a valuable role of wastewater-based epidemiology in predicting outbreaks before the onset of cases in the community.

scholarly journals Next generation sequencing of RB1gene for the molecular diagnosis of ethnic minority with retinoblastoma in Yunnan

2020 ◽  
Author(s):  
Huaiyu Gu ◽  
Zhen Zhang ◽  
Yi-shuang Xiao ◽  
Ru Shen ◽  
Hong-chao Jiang ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Next Generation Sequencing ◽  
Low Income ◽  
Eastern China ◽  
Low Income Countries ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Bioinformatics Pipeline ◽  
Generation Sequencing

Abstract Background: Retinoblastoma is a rare intraocular malignancy and typically initiated by inactivating biallelic mutations of RB1 gene. Each year, ~8,000 children worldwide are diagnosed for retinoblastoma. In high-income countries, patient survival is over 95% while low-income countries is ~30%.If disease is diagnosed early and treated in centers specializing in retinoblastoma, the survival might exceed 95% and many eyes could be safely treated and support a lifetime of good vision. In China, approximate 1,100 newly diagnosed cases are expected annually and 28 hospitals covering 25 provinces established centers classified by expertise and resources for better treatment options and follow-up. Comparing with other province of eastern China, Yunnan province is remote geographically. This might result that healthcare staff have low awareness of the role of genetic testing in management and screening in families.Methods: The patients with retinoblastoma were selected in Yunnan. DNA from blood was used for targeted gene sequencing. Then, an in-house bioinformatics pipeline was done to detect both single nucleotide variants and small insertions/deletions. The pathogenic mutations were identified and further confirmed by conventional methods and cosegregation in families.Results: Using our approach, targeted next generation sequencing was used to detect the mutation of these 12 probands. Bioinformatic predictions showed that nine mutations were found in our study and four were novel pathogenic variants in these nine mutations.Conclusions: It’s the first report to describe RB1 mutations in Yunnan children with retinoblastoma. This study would improve role of genetic testing for management and family screening.

scholarly journals Assay of frequency and spectrum of genetic variants in TTN in healthy russian population

2021 ◽  
Vol 8 (5) ◽  
pp. 29-37
Author(s):  
Yu. A. Vakhrushev ◽  
A. A. Kozyreva ◽  
S. V. Zhuk ◽  
O. P. Rotar ◽  
A. A. Kostareva
Keyword(s):  
Next Generation Sequencing ◽  
Genetic Variants ◽  
Sanger Sequencing ◽  
Heart Diseases ◽  
Genetic Research ◽  
Russian Population ◽  
Next Generation ◽  
Data Bases ◽  
International Data ◽  
Generation Sequencing

Background. Gene TTN associated with all types of cardiomyopathy, however its large size (294 b.p.) warrants a lot of individual unique genetic variants or variants with low frequency, that aggravates their interpretation. Besides that nowadays there is no data about spectrum of variants in this gene in healthy Russian population. Recognition frequency and spectrum of variants in gene TTN in healthy Russian population will allow us to use it for interpretation results of molecular genetic research for patients with different heart pathology, and define prognosis for different heart diseases.Objective. Recognize frequency and spectrum of single nucleotide and truncating variants in gene TTN in healthy Russian population and compare it with international data bases, and evaluate level of pathogenicity these variants and their distributing across titin structure.Design and methods. 192 men in age 55,8±6,6 years were tested with next-generation sequencing. Identified genetic variants were confirmed by Sanger sequencing. Results. Allele missense variant frequency (with frequency less than 0.1%) in TTN in healthy Russian population amount to 15.1 %, and truncating variants — 0.52 %. 37,9 % of them were variants of unknown significance, 62 % — likely-benign and 0.1 % — benign. There was no pathological and likely-pathological variants. Identified genetic variants distributed throughout the titin structure.Conclusion. Received result is congruent с international data bases and researches. Expended laboratory method (Next generation sequencing and confirmation with Sanger sequencing) can be used both in clinical practice, and in creating data bases of genetic variants in healthy Russian population.

scholarly journals Brain Tumor Biobank Development for Precision Medicine: Role of the Neurosurgeon

2021 ◽  
Vol 11 ◽  
Author(s):  
Emilie Darrigues ◽  
Benjamin W. Elberson ◽  
Annick De Loose ◽  
Madison P. Lee ◽  
Ebonye Green ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Next Generation Sequencing ◽  
Precision Medicine ◽  
Critical Role ◽  
First Year ◽  
Histopathological Diagnosis ◽  
Next Generation ◽  
Cancer Predisposition Syndrome ◽  
Generation Sequencing

Neuro-oncology biobanks are critical for the implementation of a precision medicine program. In this perspective, we review our first year experience of a brain tumor biobank with integrated next generation sequencing. From our experience, we describe the critical role of the neurosurgeon in diagnosis, research, and precision medicine efforts. In the first year of implementation of the biobank, 117 patients (Female: 62; Male: 55) had 125 brain tumor surgeries. 75% of patients had tumors biobanked, and 16% were of minority race/ethnicity. Tumors biobanked were as follows: diffuse gliomas (45%), brain metastases (29%), meningioma (21%), and other (5%). Among biobanked patients, 100% also had next generation sequencing. Eleven patients qualified for targeted therapy based on identification of actionable gene mutations. One patient with a hereditary cancer predisposition syndrome was also identified. An iterative quality improvement process was implemented to streamline the workflow between the operating room, pathology, and the research laboratory. Dedicated tumor bank personnel in the department of neurosurgery greatly improved standard operating procedure. Intraoperative selection and processing of tumor tissue by the neurosurgeon was integral to increasing success with cell culture assays. Currently, our institutional protocol integrates standard histopathological diagnosis, next generation sequencing, and functional assays on surgical specimens to develop precision medicine protocols for our patients. This perspective reviews the critical role of neurosurgeons in brain tumor biobank implementation and success as well as future directions for enhancing precision medicine efforts.

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