Computational protocol: Next-Generation Sequencing and In Vitro Expression Study of ADAMTS13 Single Nucleotide Variants in Deep Vein Thrombosis

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Protocol publication

[…] To investigate the potential functional effect of each SNV, the Combined Annotation Dependent Depletion (CADD;; accessed September 2016) was used. This tool integrates different functional aspects and different annotations into a single outcome, the C-score []. A scaled C-score greater or equal to 10 indicates that SNV was predicted to be the 10% most deleterious substitutions, a score greater or equal 20 indicates the 1% most deleterious. [...] Our previous study [] was limited by the size of the population enrolled. Therefore, two replication studies were carried out to validate the association between selected ADAMTS13 SNVs and the DVT risk. The ADAMTS13 SNVs which showed reduced in vitro activity levels comparable to patients’ plasma levels (p.Asp187His, p.Arg421Cys) and p.Val154Ile (patient plasma sample was missing) were selected. The other SNVs were excluded because: (i) there was an inconsistency between ex vivo and in vitro values (p.Tyr603Cys and p.Thr1249Pro) or (ii) they showed normal ex vivo and in vitro ADAMTS13 activity (p.Thr339Arg, p.Asp836Gly, p.Arg925Gly and p.His1196Gln). For the first replication study, performed in the Italian population, the allele frequency of SNVs p.Val154Ile, p.Asp187His and p.Arg421Cys was assessed in 298 patients and 298 controls from the DVT Milan study Clinical characteristics and enrolment criteria were previously described [].The targeted sequencing of the coding region, intron-exon boundaries, and 3’ and 5’ untranslated regions (UTRs) of ADAMTS13 was performed using multiplexed NGS (Human Genome Sequencing Center, Baylor College of Medicine, Houston, USA). Probes were designed by NimbleGen using genomic coordinates of our target region based on the Reference Sequence (RefSeq) collection in the UCSC Genome Browser database. Using unique barcode-sequencing tags, library pools of 8–20 samples were captured and sequenced in parallel using the Illumina HiSeq 2000 sequencing platform (Illumina, San Diego, USA). Sequence reads were processed with the Mercury analysis pipeline [] implemented in the DNAnexus platform. The presence of all variants was validated by Sanger sequencing analysis.For the second replication study, the same variants were searched in 4306 patients and 4887 controls with available DNA from the MEGA study, a large Dutch population-based case-control study, which included patients with a first event of DVT and pulmonary embolism []. The SNVs were genotyped using custom-designed TaqMan allelic discrimination assays (Applied Biosystems, Foster City, California; ) according to manufacturer’s instructions. The flow chart of the replication studies is provided ().The study was approved by the Medical Ethics Committee of the Fondazione IRCCS Ca’ Granda, Hospital Maggiore and the Ethics Committee of the Leiden University Medical Center, Leiden, The Netherlands and it has been carried out in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki). All patients were aware of the content of this study and gave a written informed consent. [...] Associations between SNVs and venous thrombosis were assessed by calculating ORs and 95% confidence intervals (CI), assuming an additive model of inheritance.The post-hoc power of the replication studies, to confirm the associations found in our previously work, was calculated using G*Power version 3.0.10 software [], based on the allele frequencies of the selected variants (p.Val154Ile, p.Asp187His and p.Arg421Cys) in the European (Non-Finnish) population reported on ExAc browser (; accessed September 2016), the overall replication sample size and assuming an OR of 2 and 0.05 two-tail alpha error. […]

Pipeline specifications

Software tools CADD, DNAnexus, G*Power
Databases UCSC Genome Browser ExAC browser
Applications Miscellaneous, WGS analysis, Genome data visualization
Organisms Homo sapiens
Diseases Venous Thrombosis