scholarly journals Next-Generation Sequencing and In Vitro Expression Study of ADAMTS13 Single Nucleotide Variants in Deep Vein Thrombosis

PLoS ONE ◽  
2016 ◽  
Vol 11 (11) ◽  
pp. e0165665 ◽  
Author(s):  
Maria Teresa Pagliari ◽  
Luca A. Lotta ◽  
Hugoline G. de Haan ◽  
Carla Valsecchi ◽  
Gloria Casoli ◽  
...  
Keyword(s):  
Deep Vein Thrombosis ◽  
Next Generation Sequencing ◽  
Single Nucleotide Variants ◽  
Expression Study ◽  
Single Nucleotide ◽  
In Vitro Expression ◽  
Vein Thrombosis ◽  
Deep Vein ◽  
Generation Sequencing

Identification of DEEP Vein Thrombosis GENETIC RISK Variants by NEXT GENERATION Sequencing of Hemostatic Genes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 710-710
Author(s):  
Luca Andrea Lotta ◽  
Mark Wang ◽  
Jin Yu ◽  
Fuli Yu ◽  
Ida Martinelli ◽  
...  
Keyword(s):  
Deep Vein Thrombosis ◽  
Next Generation Sequencing ◽  
Dna Sequencing ◽  
Binomial Probability ◽  
Next Generation ◽  
Next Generation Dna Sequencing ◽  
Vein Thrombosis ◽  
Risk Variants ◽  
Deep Vein ◽  
Generation Sequencing

Abstract Abstract 710 Next-generation DNA sequencing is opening new avenues for genetic association studies in complex diseases that, like deep vein thrombosis (DVT), have a strong genetic basis only partially accounted for by currently identified risk variants. Using next-generation DNA sequencing, we resequenced the entire protein-coding area and intron-exon boundaries of ≈200 hemostatic/pro-inflammatory genes (including all coagulation factor and anticoagulant protein genes) in 26 Italian individuals with idiopathic DVT of the lower limbs and 28 age-, gender- and ethnicity-matched healthy controls. In order to enrich for genetic component, DVT patients were selected from a cohort of 730 cases of idiopathic DVT referred to the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center (Milan, Italy) on the basis of the following criteria: (a) negative for anti-phospholipid autoantibodies, anticoagulant protein deficiencies, factor V Leiden and prothrombin G20210A, (b) age of onset <55 years. Genomic libraries with barcode sequence-tags were pooled (in pools of 8 or 16 samples) and enriched for target DNA sequences by capture on Roche NimbleGen HD2 2.1M-probe custom chips. Sequencing was performed on ABI SOLiD 4 platforms. After sequencing, reads with the barcodes were assigned to the corresponding sample and mapped to reference human genome, NCBI36/hg18, using BFAST software. We produced more than 18 gigabases of raw sequence data to sequence at high depth of coverage (>40X after duplicate read removal) the 700-kilobase target region. A total of 2351 single nucleotide variants (SNVs) and 121 short indels were identified. We developed a dedicated software in order to be able to run association analyses in our dataset using PLINK. A plain association analysis was used to compare the frequencies of common SNVs, whereas a gene-based analysis of restricted non-synonymous mutations was used to reveal potential associations of rare variants. Of 626 common SNVs tested for association, 32 SNVs from 13 genes were associated with p<0.01. These include 8 missense variants at 6 genes, 2 in genes already reported in association with DVT (FGA, ABO) and 6 in genes not previously reported (LPA, COL4A6, PLCG2 and KNG1). As a positive control we confirmed the association of rs6050 at FGA in ∼1400 Italian individuals (p=1.9 × 10−5, OR 1.45; 95% CI, 1.22–1.72). Replication of the other variants is ongoing. Gene-based analyses revealed an excess of private missense variants at anticoagulant protein genes (13 vs 3 variants; binomial probability, p=0.005) and at collagen 4 genes (14 vs 6 variants; binomial probability, p=0.02) in DVT cases compared to controls. We are currently expanding our investigations to 100 DVT cases and 100 controls. Although obtained in a relatively small number of patients and controls, our results illustrate the potential of next-generation sequencing for the discovery of genetic variation predisposing to complex diseases like DVT. Disclosures: No relevant conflicts of interest to declare.

Rare Coding Single Nucleotide Variants of ADAMTS13 Are Associated with Deep Vein Thrombosis in a Next-Generation Sequencing Association Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 107-107
Author(s):  
Luca Andrea Lotta ◽  
Giacomo Tuana ◽  
Jin Yu ◽  
Ida Martinelli ◽  
Mark Wang ◽  
...  
Keyword(s):  
Deep Vein Thrombosis ◽  
Allele Frequency ◽  
Genomic Dna ◽  
Low Frequency ◽  
Adamts13 Activity ◽  
Single Nucleotide Variants ◽  
Base Pairs ◽  
Single Nucleotide ◽  
Vein Thrombosis ◽  
Deep Vein

Abstract Abstract 107 The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Genome-wide association studies investigating hundreds of thousands of common single nucleotide variants (SNVs) identified a number of associations, but missing heritability remains. Rare (i.e. minor allele frequency below 1%) and low-frequency (i.e. minor allele frequency below 5%) SNVs of the coding area may be responsible for at least part of this missing heritability. In order to investigate this, we sequenced 700,000 base pairs of genomic DNA including the protein coding exons and intron-exon boundaries of 186 hemostatic/pro-inflammatory genes. Indexed genomic DNA libraries were co-captured on NimbleGen HD2 2.1M-probe chips and capture products were sequenced on SOLiD 4 platforms. More than 70 billion base-pairs of raw sequence data were produced to sequence the target area with a median redundancy of 45X in 94 thrombophilia-negative patients with DVT and 98 controls. Most of the 4366 SNVs identified were rare, novel and nonsynonymous indicating pathogenetic potential. We tested the association of coding SNVs in the ADAMTS13 and VWF genes, encoding two interconnected proteins with fundamental roles in hemostasis. Sequencing of the two genes yielded 109 variants, 108 SNVs and a c.8241_8442del frameshift deletion in exon 51 of VWF. Being a carrier of rare coding (prevalence in DVT: 17% [n=16]; prevalence in controls 4% [n=4]; odds ratio [OR]: 4.8; 95% confidence interval [CI]: 1.6–15.0), rare nonsynonymous (prevalence in DVT: 11% [n=10]; prevalence in controls 3% [n=3]; OR: 3.8; 95% CI: 1.0–14.2) or low-frequency coding (prevalence in DVT: 36% [n=34]; prevalence in controls 23% [n=23]; OR: 1.9; 95% CI: 1.0–3.5) SNVs of ADAMTS13 was associated with DVT. Carrying rare or low-frequency SNVs of VWF was not associated with DVT. The 11 different rare missense variants of ADAMTS13 found in DVT patients had never been described in association with congenital thrombotic thrombocytopenic purpura. Patients carrying at least one ADAMTS13 mutation of the categories associated with DVT had lower plasmatic levels of ADAMTS13 activity compared to patients without mutations. The change in ADAMTS13 activity was −7% (95% CI: −24 to 10%) for patients with rare coding ADAMTS13 mutations, −12% (95% CI: −30 to 6%) for patients with rare nonsynonymous mutations and −29% (95% CI: −52 to −6%) for patients with missense mutations predicted to be damaging for protein function by SIFT. Our results uncover for the first time a link between ADAMTS13, an important regulator of hemostasis implicated in microvascular thrombosis, and DVT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Model Study of In Silico Proficiency Testing for Clinical Next-Generation Sequencing

2016 ◽  
Vol 140 (10) ◽  
pp. 1085-1091 ◽  
Author(s):  
Eric J. Duncavage ◽  
Haley J. Abel ◽  
Jason D. Merker ◽  
John B. Bodner ◽  
Qin Zhao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Proficiency Testing ◽  
In Silico ◽  
Absolute Difference ◽  
Ion Torrent ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Clinical Laboratories ◽  
Generation Sequencing

Context.—Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample. Objective.—To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically. Design.—DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. Results.—Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. Conclusions.—The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.

Genome study of PDGFRA D842V mutant GIST using next generation sequencing approach.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 10540-10540
Author(s):  
Maria A. Pantaleo ◽  
Annalisa Astolfi ◽  
Milena Urbini ◽  
Valentina Indio ◽  
Margherita Nannini ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Tyrosine Kinases ◽  
Therapeutic Targets ◽  
Mapk Signaling ◽  
Gastric Gist ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Generation Sequencing

10540 Background: Mutations of the receptors KIT and PDGFRA in GIST are the oncogenetic events of disease as well as the targets of molecular therapies. Within the PDGFRA mutations, the D842V mutation in exon 18 confers in vitro and in vivo resistance to imatinib. Next generation sequencing techniques may completely dissect all the possible somatic mutations and genomic rearrangements in order to identify novel therapeutic targets in this patients population. Methods: Five patients with gastric GIST were analyased (3 M, 2 F; mean age 65,5 years, range 51-77). The tumor dimension ranged between 3 and 15 cm, MI < 2 and 8 /50 HPF. No metastases were present in all cases. The KIT and PDGFRA anlysis showed a D842V mutation in exon 18 of PDGFRA in all cases. Whole transcriptome sequencing was performed with Illumina HiScanSQ platform with a paired-end strategy (75x2). After performing quality controls, the short reads were mapped with Tophat-Botie pipeline against the human reference genome (HG19). The variations, such as Single nucleotide variants (SNVs) and InDels, were called by SNVMix2 (a software suited for SNVs discovery in tumor samples) implementing an accurate filtering procedures developed in our laboratory. Two predictors of mutations effect at protein level (SNPs&GO and Provean) were employed in order to prioritize the emerging variation. Results: An average of 206 coding non-synonymous variants were highlighted in the five GIST samples, of which ~ 30% were predicted as deleterious with at least one predictor. In addition to PDGFRA D842V mutation, in all five patients were found mutations on different receptor tyrosine kinases, such as FGFR4 and DDR2. Moreover three out of five patients harboured mutations on members of the MDR/TAP subfamily that are involved in multidrug resistance, in particular on ABCB1, ABCB4 and on ABCB6 genes. Other mutations were found on the hedgehog and MAPK signaling pathway and on SNF/SWI chromatin remodeling complex. Conclusions: New molecular events have been identified in PDGFRA D842V mutant GIST. These data should be validated in larger series and the role of these mutations as therapeutic targets should be further investigated.

scholarly journals Prostate cancer heterogeneity assessment with multi-regional sampling and alignment-free methods

2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Ross G Murphy ◽  
Aideen C Roddy ◽  
Shambhavi Srivastava ◽  
Esther Baena ◽  
David J Waugh ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Cancer Heterogeneity ◽  
Alignment Free ◽  
Treatment Indications ◽  
Patient Heterogeneity ◽  
Genomic Locations ◽  
Generation Sequencing

Abstract Combining alignment-free methods for phylogenetic analysis with multi-regional sampling using next-generation sequencing can provide an assessment of intra-patient tumour heterogeneity. From multi-regional sampling divergent branching, we validated two different lesions within a patient’s prostate. Where multi-regional sampling has not been used, a single sample from one of these areas could misguide as to which drugs or therapies would best benefit this patient, due to the fact these tumours appear to be genetically different. This application has the power to render, in a fraction of the time used by other approaches, intra-patient heterogeneity and decipher aberrant biomarkers. Another alignment-free method for calling single-nucleotide variants from raw next-generation sequencing samples has determined possible variants and genomic locations that may be able to characterize the differences between the two main branching patterns. Alignment-free approaches have been applied to relevant clinical multi-regional samples and may be considered as a valuable option for comparing and determining heterogeneity to help deliver personalized medicine through more robust efforts in identifying targetable pathways and therapeutic strategies. Our study highlights the application these tools could have on patient-aligned treatment indications.

Microrna-342-3p Loaded by Human Umbilical Cord Mesenchymal Stem Cells-derived Exosomes Attenuates Deep Vein Thrombosis by Downregulating EDNRA

2021 ◽  
Author(s):  
Zhiyu Pan ◽  
Qian Chen ◽  
Hongjian Ding ◽  
Huaqing Li
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Deep Vein Thrombosis ◽  
Umbilical Cord ◽  
Umbilical Vein ◽  
Dynamic Balance ◽  
Human Umbilical Cord ◽  
Vein Thrombosis ◽  
Deep Vein

Abstract Objectivexosomes (exos) exert biological functions to maintain the dynamic balance of cells and tissues by transferring biological cargo to recipient cells. Thus, this study explored human umbilical cord mesenchymal stem cells (hucMSCs)-derived exo transfer of microRNA (miR)-342-3p in deep vein thrombosis (DVT).MethodsHucMSCs were isolated and transfected with miR-342-3p antagomir/agomir. Then, hucMSCs-exos were extracted and injected into rats with DVT to observe inflammation and pathological damage in thrombotic vein. HucMSCs-exos were co-cultured with human umbilical vein endothelial cells (HUVECs) in vitro to observe angiogenesis. miR-342-3p and endothelin A receptor (EDNRA) expression in rats with DVT, as well as their interaction was analyzed.ResultsmiR-342-3p was downregulated and EDNRA was upregulated in rats with DVT. HucMSCs-exos induced the repair of venous thrombosis and suppressed inflammation and pathological injury in rats with DVT, as well as promoted angiogenesis of HUVECs. Upregulated miR-342-3p delivery by hucMSCs-exos alleviated DVT in rats and improved angiogenesis of HUVECs, while downregulated miR-342-3p performed oppositely. miR-342-3p targeted EDNRA, and the effect of hucMSCs-exos transfer of upregulated miR-342-3p was rescued by overexpressing EDNRA.ConclusionBriefly, miR-342-3p loaded by hucMSCs-exos attenuates DVT by downregulating EDNRA, offering a novel direction to treat DVT.

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