The TS Zimmerman Program for the Molecular and Clinical Biology of von Willenbrand disease (VWD) is a multinational Program Project established to further the study of VWD in the United States and to contrast these studies with the studies initiated previously in the EU and Canada. In order to gain further insight into the clinical expression and penetrance of established types of VWD, we performed full gene DNA sequence analysis on VWD patients and normal controls. Previously, we reported new sequence variations identified from 50 VWD index cases and 113 normal controls in our study. This is an updated report of the sequence variations identified in a second cohort of 44 index cases with type 1, 2 and 3 VWD and 48 normal controls. Fourteen of these index cases have known mutations, 3 of which also have a second new mutation. Seven additional index cases had 1 or 2 new mutations. Three cases had new polymorphisms identified in our first cohort. Thirteen new mutations were identified in type 1 and type 3 patients including 3 nonsense mutations, 2 insertions, and 8 missense mutations. In cases where mutations were identified, 48% of the identified mutations were new mutations that have not been reported in the Sheffield VWF Mutation Database. A similar frequency of new mutation was observed in our first cohort (46%). In 20 of 44 (45%) patients with either type 1 or type 3 VWD, no sequence variations were identified in the VWF coding region. In our previous cohort, sequence variations were not detected in 30% of patients. Mutations in the non-coding region of the gene or mutations not detectable by DNA sequencing have not been ruled out in this group of patients. Since VWF polymorphisms are not well characterized in all exons and in different ethnic groups, full VWF laboratory testing and gene sequencing of over 160 normal controls was completed in our study. In our first report of the 113 normal controls, we identified 19 new sequence variations that were found mainly in African Americans. In the second cohort of 48 normal controls, we identified 2 new sequence variations (1087C>T and 1463C>G). The decreasing number of new sequence variations in the normal controls is in contrast to the index cases where a similar percent of new mutations were detected in both cohorts and may indicate that the majority of polymorphisms have been identified. Seven of 19 new sequence variations seen in the first cohort of normal controls were also found in the second cohort further supporting that they are polymorphisms. Previously, we identified six sequence variations that were previously reported as VWF mutations. In this study, the same six sequence variations (2220G>A, 2451T>A, 2771G>A, 3686T>G, 3692A>C and 6859C>T) were detected in the normal controls providing further evidence that these sequence variations are most likely polymorphisms. In addition, we detected two other reported mutations (6187C>T; P2063S and 2561G>A; R854Q) each in two normal controls. Our data indicate that sequencing of large numbers of normal controls is important to aid in differentiating mutations from polymorphisms. This study emphasizes the importance of understanding the ethnic-specific sequence variations in African Americans such that polymorphisms are not misidentified as mutations. Our data also suggest that the genetic variation in the VWF gene is extensive and that many low frequency mutations and polymorphisms remain to be identified. Differentiating polymorphisms from disease-causing sequence variations that affect the diagnosis of VWD and/or hemorrhagic risk is important but continues to be challenging in this bleeding disorder.

Disclosures: Montgomery:Baxter: Consultancy; GTI Diagnostics: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding.

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