Detection of VWF Copy Number Variations by Microarray Analysis in Von Willebrand Disease Type 3 Index Cases

Abstract 380^FN2

As part of the TS Zimmerman Program for the Molecular and Clinical Biology of Von Willebrand disease (VWD), we performed complete laboratory phenotyping and full gene DNA sequence analysis on VWD index cases. VWD type 3 is an autosomal recessive disorder characterized by undetectable levels of von Willebrand factor (VWF). These patients are expected to have VWF mutations that abolish expression of the protein on both VWF alleles. We identified a group of VWD type 3 patients in which one or both alleles did not have a VWF mutation identified by DNA sequence analysis. Although DNA sequencing is considered to be the gold standard for mutation detection, this technique does not detect copy number variations (CNVs). Microarrays have been successfully used to detect CNVs missed by DNA sequencing in other disorders such as Duchenne muscular dystrophy. In order to investigate the role of VWF deletion and duplication in VWD, we designed a targeted gene high-resolution oligonucleotide comparative genomic hybridization (CGH) array on an Oxford Gene Technologies (OGT) 60K platform that permits high resolution analysis of the VWF gene using long oligonucleotides probes (∼45–60mer). The average spacing was 65 bp within coding regions and 178 bp within intronic regions. The advantages of CGH array over techniques such as multiplex ligation-dependent probe amplification (MLPA) is that many probes interrogate each region so detection is less likely to be affected by sequence variants and polymorphisms. In addition, the intronic oligonucleotide probes allowed us to detect dosage changes within the entire genomic region of the gene and determine the approximate breakpoint location. From the approximate breakpoints position, PCR primers can be designed to confirm the copy number variant and identify the exact breakpoint by sequencing. We analyzed a total of fourteen type 3 patients using the VWF CGH array. We identified deletions in the VWF gene in four patients. Three of the patients also had new mutations detected by DNA sequencing.

In patients 1 and 2 we identified an in-frame deletion of exons 4–5. This deletion was previously reported as recurrent mutation in type 1 and type 3 patients by Sutherland et al, Blood (2009) 114:1091. Patient 2 also had a novel deletion in exons 1–3. This deletion would remove the initiating ATG in exon 2. In patient 3, we detected an out of frame deletion of exon 18. Exon 18 contains the propeptide cleavage site. Finally, in patient 4 we detected an out of frame deletion of exons 35–38. These deletions are expected to be type 3 alleles. The breakpoints of each deletion are intronic. PCR will confirm the variant and identify the breakpoint. Our study demonstrates that deletions in the VWF gene may be a common mutation mechanism. We detected deletions in 29% of type 3 patients that did not have two VWF mutations identified by sequencing. CGH arrays are an effective way of identifying deletion and duplication in the VWF gene and mapping their approximate breakpoint locations.