High-throughput molecular diagnosis of von Willebrand disease by next generation sequencing methods.
Genetic analysis of von Willebrand disease by von Willebrand factor gene sequencing has not yet become routine practice. Nevertheless, the prospects for molecular diagnosis have changed dramatically in recent years with the unveiling of next-generation sequencing platforms. With the goal of applying this technology to von Willebrand disease, we designed a strategy for von Willebrand factor gene enrichment and multiplexing based on short polymerase chain reactions. Forty patients were simultaneously analyzed enabling the identification of 43 mutations, including 36 substitutions, 2 intronic splice site mutations, 2 indels, and 3 deletions. By pooling patient genomic DNA before polymerase chain reaction enrichment, indexing samples with barcode tags, and re-sequencing on the next-generation sequencing instrument, at least 350 patients and relatives per run can be simultaneously analyzed in a fast, inexpensive manner. This is one of the first reports in which this technology has been shown to be feasible for large-scale mutation screening by single gene re-sequencing.
Unitat de Diagnòstic i Teràpia Molecular, Banc de Sang i Teixits, Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain.
Irene
I
Corrales
Susana
S
Catarino
Júlia
J
Ayats
David
D
Arteta
Carmen
C
Altisent
Rafael
R
Parra
Francisco
F
Vidal
D005820:Genetic Testing; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D017422:Sequence Analysis, DNA / Q000379:methods*; D014842:von Willebrand Diseases / Q000175:diagnosis / Q000235:genetics; D014841:von Willebrand Factor / Q000235:genetics*
D014841:von Willebrand Factor