Identification of Copy Number Variants in Type 1 and Type 3 VWD by aCGH in the Zimmerman Program

von Willebrand Disease (VWD) is a common inherited bleeding disorder caused by a quantitative or qualitative abnormality of von Willebrand Factor (VWF). Genetic analysis for VWD commonly utilizes DNA sequencing to identify sequence variants (SV) in VWF, however this technique cannot detect copy number variants (CNV), including deletions and duplications, which cause approximately 10% of genetic diseases. To determine the role of CNV in VWD, we performed array Comparative Genomic Hybridization (aCGH) on 122 mutation-negative subjects enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD (Zimmerman Program) where full-length VWF sequencing, including 5' and 3' regions and intron/exon boundaries, failed to identify a SV that would explain their phenotype. These subjects included 84 low VWF, 16 type 1, 10 type 1C, 2 type 2A, and 10 type 3. Clinical testing performed at BloodCenter of Wisconsin included FVIII, VWF:Ag, VWF:RCo and VWFpp. Bleeding symptoms were quantified using the ISTH bleeding score (BS). A VWF high-resolution aCGH array was custom designed using long oligonucleotide probes ~60 bp in length to avoid multiple sequence variations, repeated sequences and the pseudogene. Data was manually inspected and analyzed with CytoSure Interpret Software. A multiplex PCR approach was used to map deletion breakpoints and to confirm the presence and zygosity of the deletions in the index case as well as family members (FM).

Large deletions were identified in 12% of this cohort (14 subjects) where no other VWF mutations were found. Of these 14 subjects, 5 were type 1, 5 type 1C and 4 type 3, which represents CNV found in 31% of the type 1, 50% of type 1C and 40% of the type 3 tested. No CNV were found in the 84 low VWF subjects or the 2 type 2A patients. Mean VWF:Ag (6 IU/dl) and VWF:RCo (13 IU/dl) in subjects with deletions was statistically different (p<0.0001) compared to those with no large deletion (31 and 33 IU/dl). Subjects with deletions had a median ISTH BS of 4 whereas those with no deletion had a score of 6 which was not significantly different (p=0.61).

The most common deletion was the previously described in-frame exon 4-5 (c.221-977_532+7059del) which was identified in 5% of this cohort (4 type 1 subjects and 2 type 3 subjects). In these 6 unrelated families, the FM with the deletion had a lower mean VWF:Ag (25 IU/dl), VWF:RCo (26 IU/dl) and higher ISTH BS (3) than FM without the deletion with VWF:Ag of 74, VWF:RCo of 76 and ISTH BS of 1 (p<0.05). The second most frequent (4%) was an in-frame deletion, exon 33-34 (c.5621-478_5842+2440delinsGCAGCATAAGCATAAAGC) which was the only CNV found in the type 1C group (5/10 subjects). FM with exon 33-34 deletion had mean VWF:Ag of 21, increased VWFpp/VWF:Ag (4.3) and ISTH BS (3), while FM without the deletion had mean VWF:Ag of 107, normal VWFpp/VWF:Ag (1.3) and ISTH BS (1). This 3.5kb deletion results in a loss of 9 cysteines which may be associated with reduced plasma survival of the VWF in these patients. The remaining deletions were found in single subjects, representing 1% of the index cases tested. We identified novel deletions in exon 1-3 and exon 11. Interestingly, the exon 1-3 deletion had a novel breakpoint, c.-30029_220+3487del , and was found in a type 3 index case who was compound heterozygote for the exon 4-5 deletion as well. An out-of-frame deletion, c.2282-809_2442+2811delinsT, was found in exon 18 in 1 type 3 subject. This deletion was reported in the VWF database but the breakpoints had not been mapped. This deletion alone causes low VWF and confers a type 3 phenotype when inherited with a single nucleotide deletion on the other VWF allele. Another novel out-of-frame deletion, exon 35-38 (c.5843-2754_6799-1517del), was found in a type 3 patient where we confirmed that it segregated with FM who had type 1 VWD, but resulted in type 3 when inherited along with a frameshift mutation caused by a single nucleotide duplication.

aCGH is a sensitive and powerful method to identify CNV in the VWF gene, narrow the breakpoint locations for mapping, and characterize the different mechanisms for type 1 and type 3 VWD. Both in-frame and out-of-frame deletions may be a common mechanism of type 1 and result in reduced levels of VWF in plasma. While aCGH may not be as beneficial for subjects with "low VWF", it should be considered when attempting to determine the underlying genetic cause of type 1 patients with VWF:Ag levels <30 or type 3 when a SV is not identified by VWF sequencing.