Dataset Information

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

ABSTRACT:

Background

Deep vein thrombosis (DVT) genetic predisposition is partially known.

Objectives

This study aimed at assessing the functional impact of nine ADAMTS13 single nucleotide variants (SNVs) previously reported to be associated as a group with DVT in a burden test and the individual association of selected variants with DVT risk in two replication studies.

Methods

Wild-type and mutant recombinant ADAMTS13 were transiently expressed in HEK293 cells. Antigen and activity of recombinant ADAMTS13 were measured by ELISA and FRETS-VWF73 assays, respectively. The replication studies were performed in an Italian case-control study (Milan study; 298/298 patients/controls) using a next-generation sequencing approach and in a Dutch case-control study (MEGA study; 4306/4887 patients/controls) by TaqMan assays.

Results

In vitro results showed reduced ADAMTS13 activity for three SNVs (p.Val154Ile [15%; 95% confidence interval [CI] 14-16], p.Asp187His [19%; 95%[CI] 17-21], p.Arg421Cys [24%; 95%[CI] 22-26]) similar to reduced plasma ADAMTS13 levels of patients carriers for these SNVs. Therefore these three SNVs were interrogated for risk association. The first replication study identified 3 heterozygous carriers (2 cases, 1 control) of p.Arg421Cys (odds ratio [OR] 2, 95%[CI] 0.18-22.25). The second replication study identified 2 heterozygous carriers (1 case, 1 control) of p.Asp187His ([OR] 1.14, 95%[CI] 0.07-18.15) and 10 heterozygous carriers (4 cases, 6 controls) of p.Arg421Cys ([OR] 0.76, 95%[CI] 0.21-2.68).

Conclusions

Three SNVs (p.Val154Ile, p.Asp187His and p.Arg421Cys) showed reduced ex vivo and in vitro ADAMTS13 levels. However, the low frequency of these variants makes it difficult to confirm their association with DVT.

SUBMITTER: Pagliari MT

PROVIDER: S-EPMC5089687 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

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

Pagliari Maria Teresa MT Lotta Luca A LA de Haan Hugoline G HG Valsecchi Carla C Casoli Gloria G Pontiggia Silvia S Martinelli Ida I Passamonti Serena M SM Rosendaal Frits R FR Peyvandi Flora F

PloS one 20161101 11

<h4>Background</h4>Deep vein thrombosis (DVT) genetic predisposition is partially known.<h4>Objectives</h4>This study aimed at assessing the functional impact of nine ADAMTS13 single nucleotide variants (SNVs) previously reported to be associated as a group with DVT in a burden test and the individual association of selected variants with DVT risk in two replication studies.<h4>Methods</h4>Wild-type and mutant recombinant ADAMTS13 were transiently expressed in HEK293 cells. Antigen and activity ...[more]

PMID: 27802307

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Project description:Next-generation DNA sequencing is opening new avenues for genetic association studies in common diseases that, like deep vein thrombosis (DVT), have a strong genetic predisposition still largely unexplained by currently identified risk variants. In order to develop sequencing and analytical pipelines for the application of next-generation sequencing to complex diseases, we conducted a pilot study sequencing the coding area of 186 hemostatic/proinflammatory genes in 10 Italian cases of idiopathic DVT and 12 healthy controls.A molecular-barcoding strategy was used to multiplex DNA target capture and sequencing, while retaining individual sequence information. Genomic libraries with barcode sequence-tags were pooled (in pools of 8 or 16 samples) and enriched for target DNA sequences. Sequencing was performed on ABI SOLiD-4 platforms. We produced > 12 gigabases of raw sequence data to sequence at high coverage (average: 42X) the 700-kilobase target area in 22 individuals. A total of 1876 high-quality genetic variants were identified (1778 single nucleotide substitutions and 98 insertions/deletions). Annotation on databases of genetic variation and human disease mutations revealed several novel, potentially deleterious mutations. We tested 576 common variants in a case-control association analysis, carrying the top-5 associations over to replication in up to 719 DVT cases and 719 controls. We also conducted an analysis of the burden of nonsynonymous variants in coagulation factor and anticoagulant genes. We found an excess of rare missense mutations in anticoagulant genes in DVT cases compared to controls and an association for a missense polymorphism of FGA (rs6050; p = 1.9 × 10(-5), OR 1.45; 95% CI, 1.22-1.72; after replication in > 1400 individuals).We implemented a barcode-based strategy to efficiently multiplex sequencing of hundreds of candidate genes in several individuals. In the relatively small dataset of our pilot study we were able to identify bona fide associations with DVT. Our study illustrates the potential of next-generation sequencing for the discovery of genetic variation predisposing to complex diseases.

Project description:BackgroundWe previously described the association between rare ADAMTS13 single nucleotide variants (SNVs) and deep vein thrombosis (DVT). Moreover, DVT patients with at least one rare ADAMTS13 SNV had a lower ADAMTS13 activity than non-carriers.AimsTo confirm ADAMTS13 variants association with DVT and reduced plasma ADAMTS13 activity levels in a larger population. To investigate the role of VWF and F8 variants.MethodsADAMTS13, VWF and F8 were sequenced using next-generation sequencing in 594 Italian DVT patients and 571 controls. Genetic association testing was performed using logistic regression and gene-based tests. The association between rare ADAMTS13 variants and the respective plasmatic activity, available for 365 cases and 292 controls, was determined using linear regression. All analyses were age-, sex- adjusted.ResultsWe identified 48 low-frequency/common and 272 rare variants. Nine low-frequency/common variants had a P<0.05, but a false discovery rate between 0.06 and 0.24. Of them, 7 were found in ADAMTS13 (rs28641026, rs28503257, rs685523, rs3124768, rs3118667, rs739469, rs3124767; all protective) and 2 in VWF (rs1800382 [risk], rs7962217 [protective]). Rare ADAMTS13 variants were significantly associated with DVT using the burden, variable threshold (VT) and UNIQ (P<0.05), but not with C-ALPHA, SKAT and SKAT-O tests. Rare VWF and F8 variants were not associated with DVT. Carriers of rare ADAMTS13 variants had lower ADAMTS13 activity than non-carriers (ß -6.2, 95%CI -11,-1.5). This association was stronger for DVT patients than controls (ß -7.5, 95%CI -13.5,-1.5 vs. ß -2.9, 95%CI -10.4,4.5).ConclusionsADAMTS13 and VWF low-frequency/common variants mainly showed a protective effect, although their association with DVT was not confirmed. DVT patients carrying a rare ADAMTS13 variants had slightly reduced ADAMTS13 activity levels, but a higher DVT risk. Rare VWF and FVIII variants were not associated with DVT suggesting that other mechanisms are responsible for the high VWF and FVIII levels measured in DVT patients.

Dataset Information

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

Background

Objectives

Methods

Results

Conclusions

Publications

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

Similar Datasets

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