Identification of genetic risk variants for deep vein thrombosis by multiplexed next-generation sequencing of 186 hemostatic/pro-inflammatory genes

Luca A. Lotta, Mark Wang, Jin Yu, Ida Martinelli, Fuli Yu, Serena M. Passamonti, Dario Consonni, Emanuela Pappalardo, Marzia Menegatti, Steven E. Scherer, Lora L. Lewis, Humeira Akbar, Yuanqing Wu, Matthew N. Bainbridge, Donna M. Muzny, Pier M. Mannucci, Richard A. Gibbs, Flora Peyvandi

Research output: Contribution to journalArticle

Abstract

Background: 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. Results: 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). Conclusions: 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.

Original languageEnglish
Article number7
JournalBMC Medical Genomics
Volume5
DOIs
Publication statusPublished - 2012

Fingerprint

Hemostatics
Venous Thrombosis
Genes
Anticoagulants
Mutation
Blood Coagulation Factors
Genomic Library
Genetic Association Studies
Missense Mutation
Genetic Predisposition to Disease
DNA Sequence Analysis
Nucleotides
Databases
DNA

Keywords

  • Deep vein thrombosis
  • DVT
  • FGA
  • heamostateome
  • multiplexing
  • next-generation sequencing
  • rs6025
  • target capture
  • venous thromboembolism
  • VTE

ASJC Scopus subject areas

  • Genetics(clinical)
  • Genetics

Cite this

Identification of genetic risk variants for deep vein thrombosis by multiplexed next-generation sequencing of 186 hemostatic/pro-inflammatory genes. / Lotta, Luca A.; Wang, Mark; Yu, Jin; Martinelli, Ida; Yu, Fuli; Passamonti, Serena M.; Consonni, Dario; Pappalardo, Emanuela; Menegatti, Marzia; Scherer, Steven E.; Lewis, Lora L.; Akbar, Humeira; Wu, Yuanqing; Bainbridge, Matthew N.; Muzny, Donna M.; Mannucci, Pier M.; Gibbs, Richard A.; Peyvandi, Flora.

In: BMC Medical Genomics, Vol. 5, 7, 2012.

Research output: Contribution to journalArticle

Lotta, Luca A. ; Wang, Mark ; Yu, Jin ; Martinelli, Ida ; Yu, Fuli ; Passamonti, Serena M. ; Consonni, Dario ; Pappalardo, Emanuela ; Menegatti, Marzia ; Scherer, Steven E. ; Lewis, Lora L. ; Akbar, Humeira ; Wu, Yuanqing ; Bainbridge, Matthew N. ; Muzny, Donna M. ; Mannucci, Pier M. ; Gibbs, Richard A. ; Peyvandi, Flora. / Identification of genetic risk variants for deep vein thrombosis by multiplexed next-generation sequencing of 186 hemostatic/pro-inflammatory genes. In: BMC Medical Genomics. 2012 ; Vol. 5.
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T1 - Identification of genetic risk variants for deep vein thrombosis by multiplexed next-generation sequencing of 186 hemostatic/pro-inflammatory genes

AU - Lotta, Luca A.

AU - Wang, Mark

AU - Yu, Jin

AU - Martinelli, Ida

AU - Yu, Fuli

AU - Passamonti, Serena M.

AU - Consonni, Dario

AU - Pappalardo, Emanuela

AU - Menegatti, Marzia

AU - Scherer, Steven E.

AU - Lewis, Lora L.

AU - Akbar, Humeira

AU - Wu, Yuanqing

AU - Bainbridge, Matthew N.

AU - Muzny, Donna M.

AU - Mannucci, Pier M.

AU - Gibbs, Richard A.

AU - Peyvandi, Flora

PY - 2012

Y1 - 2012

N2 - Background: 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. Results: 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). Conclusions: 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.

AB - Background: 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. Results: 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). Conclusions: 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.

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KW - VTE

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