title: Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys-Dietz syndromes.
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.
authors.affiliation: Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.
authors.forename: Machteld
authors.initials: M
authors.lastname: Baetens
authors.forename: Lut
authors.initials: L
authors.lastname: Van Laer
authors.forename: Kim
authors.initials: K
authors.lastname: De Leeneer
authors.forename: Jan
authors.initials: J
authors.lastname: Hellemans
authors.forename: Joachim
authors.initials: J
authors.lastname: De Schrijver
authors.forename: Hendrik
authors.initials: H
authors.lastname: Van De Voorde
authors.forename: Marjolijn
authors.initials: M
authors.lastname: Renard
authors.forename: Hal
authors.initials: H
authors.lastname: Dietz
authors.forename: Ronald V
authors.initials: RV
authors.lastname: Lacro
authors.forename: Björn
authors.initials: B
authors.lastname: Menten
authors.forename: Wim
authors.initials: W
authors.lastname: Van Criekinge
authors.forename: Julie
authors.initials: J
authors.lastname: De Backer
authors.forename: Anne
authors.initials: A
authors.lastname: De Paepe
authors.forename: Bart
authors.initials: B
authors.lastname: Loeys
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Coucke
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D055947:Loeys-Dietz Syndrome / Q000175:diagnosis* / Q000235:genetics*; D008382:Marfan Syndrome / Q000175:diagnosis* / Q000235:genetics*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010865:Pilot Projects; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers