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Applying massive parallel sequencing to molecular diagnosis of Marfan and Loeys‐Dietz syndromes
Author(s) -
Baetens Machteld,
Van Laer Lut,
De Leeneer Kim,
Hellemans Jan,
De Schrijver Joachim,
Van De Voorde Hendrik,
Renard Marjolijn,
Dietz Hal,
Lacro Ronald V.,
Menten Björn,
Van Criekinge Wim,
De Backer Julie,
De Paepe Anne,
Loeys Bart,
Coucke Paul J.
Publication year - 2011
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.21525
Subject(s) - multiplex ligation dependent probe amplification , sanger sequencing , biology , genetics , fibrillin , missense mutation , multiplex , exon , mutation , marfan syndrome , dna sequencing , gene , bioinformatics , computational biology , medicine
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. Hum Mutat 32:1–10, 2011. © 2011 Wiley‐Liss, Inc.