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.