Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes

Machteld Baetens, Lut Van Laer, Kim De Leeneer, Jan Hellemans, Joachim De Schrijver, Hendrik Van De Voorde, Marjolijn Renard, Harry C Dietz, Ronald V. Lacro, Björn Menten, Wim Van Criekinge, Julie De Backer, Anne De Paepe, Bart Loeys, Paul J. Coucke

Research output: Contribution to journalArticle

Abstract

The Marfan (MFS) and Loeys-Dietz (LDS) syndromes are caused by mutations in the fibrillin-1 (FBN1) and Transforming Growth Factor Beta Receptor 1 and 2 (TGFBR1 and TGFBR2) genes, respectively. With the current conventional mutation screening technologies, analysis of this set of genes is time consuming and expensive. We have tailored a cost-effective and reliable mutation discovery strategy using multiplex PCR followed by Next Generation Sequencing (NGS). In a first stage, genomic DNA from five MFS or LDS patient samples with previously identified mutations and/or polymorphisms in FBN1 and TGFBR1 and 2 were analyzed and revealed all expected variants. In a second stage, we validated the technique on 87 samples from MFS patients fulfilling the Ghent criteria. This resulted in the identification of 75 FBN1 mutations, of which 67 were unique. Subsequent Multiplex Ligation-dependent Probe Amplification (MLPA) analysis of the remaining negative samples identified four large deletions/insertions. Finally, Sanger sequencing identified a missense mutation in FBN1 exon 1 that was not included in the NGS workflow. In total, there was an overall mutation identification rate of 92%, which is in agreement with data published previously. We conclude that multiplex PCR of all coding exons of FBN1 and TGFBR1/2 followed by NGS analysis and MLPA is a robust strategy for time- and cost-effective identification of mutations.

Original languageEnglish (US)
Pages (from-to)1053-1062
Number of pages10
JournalHuman Mutation
Volume32
Issue number9
DOIs
StatePublished - Sep 2011

Fingerprint

Loeys-Dietz Syndrome
Multiplex Polymerase Chain Reaction
Mutation
Exons
Costs and Cost Analysis
Workflow
Mutation Rate
Missense Mutation
Genes
Fibrillin-1
Technology
DNA

Keywords

  • FBN1
  • Loeys-Dietz
  • Marfan
  • Massive parallel sequencing
  • Multiplex PCR
  • TGFBR1
  • TGFBR2

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Baetens, M., Van Laer, L., De Leeneer, K., Hellemans, J., De Schrijver, J., Van De Voorde, H., ... Coucke, P. J. (2011). Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes. Human Mutation, 32(9), 1053-1062. https://doi.org/10.1002/humu.21525

Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes. / Baetens, Machteld; Van Laer, Lut; De Leeneer, Kim; Hellemans, Jan; De Schrijver, Joachim; Van De Voorde, Hendrik; Renard, Marjolijn; Dietz, Harry C; Lacro, Ronald V.; Menten, Björn; Van Criekinge, Wim; De Backer, Julie; De Paepe, Anne; Loeys, Bart; Coucke, Paul J.

In: Human Mutation, Vol. 32, No. 9, 09.2011, p. 1053-1062.

Research output: Contribution to journalArticle

Baetens, M, Van Laer, L, De Leeneer, K, Hellemans, J, De Schrijver, J, Van De Voorde, H, Renard, M, Dietz, HC, Lacro, RV, Menten, B, Van Criekinge, W, De Backer, J, De Paepe, A, Loeys, B & Coucke, PJ 2011, 'Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes', Human Mutation, vol. 32, no. 9, pp. 1053-1062. https://doi.org/10.1002/humu.21525
Baetens M, Van Laer L, De Leeneer K, Hellemans J, De Schrijver J, Van De Voorde H et al. Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes. Human Mutation. 2011 Sep;32(9):1053-1062. https://doi.org/10.1002/humu.21525
Baetens, Machteld ; Van Laer, Lut ; De Leeneer, Kim ; Hellemans, Jan ; De Schrijver, Joachim ; Van De Voorde, Hendrik ; Renard, Marjolijn ; Dietz, Harry C ; Lacro, Ronald V. ; Menten, Björn ; Van Criekinge, Wim ; De Backer, Julie ; De Paepe, Anne ; Loeys, Bart ; Coucke, Paul J. / Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes. In: Human Mutation. 2011 ; Vol. 32, No. 9. pp. 1053-1062.
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AU - Renard, Marjolijn

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AU - Lacro, Ronald V.

AU - Menten, Björn

AU - Van Criekinge, Wim

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N2 - The Marfan (MFS) and Loeys-Dietz (LDS) syndromes are caused by mutations in the fibrillin-1 (FBN1) and Transforming Growth Factor Beta Receptor 1 and 2 (TGFBR1 and TGFBR2) genes, respectively. With the current conventional mutation screening technologies, analysis of this set of genes is time consuming and expensive. We have tailored a cost-effective and reliable mutation discovery strategy using multiplex PCR followed by Next Generation Sequencing (NGS). In a first stage, genomic DNA from five MFS or LDS patient samples with previously identified mutations and/or polymorphisms in FBN1 and TGFBR1 and 2 were analyzed and revealed all expected variants. In a second stage, we validated the technique on 87 samples from MFS patients fulfilling the Ghent criteria. This resulted in the identification of 75 FBN1 mutations, of which 67 were unique. Subsequent Multiplex Ligation-dependent Probe Amplification (MLPA) analysis of the remaining negative samples identified four large deletions/insertions. Finally, Sanger sequencing identified a missense mutation in FBN1 exon 1 that was not included in the NGS workflow. In total, there was an overall mutation identification rate of 92%, which is in agreement with data published previously. We conclude that multiplex PCR of all coding exons of FBN1 and TGFBR1/2 followed by NGS analysis and MLPA is a robust strategy for time- and cost-effective identification of mutations.

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