ALBERT

Description: Introduction von Willebrand disease (VWD) is a common inherited bleeding disorder with a reported incidence ranging from 0.01% to 1%. VWD is classified as quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). Classical VWD type 2A, the most common qualitative defect of VWD, is characterized by decreased VWF-dependent platelet adhesion and a selective deficiency of high molecular weight VWF multimers. The majority of type 2A VWD is inherited in an autosomal dominant fashion. This subtype is caused by missense variants acting through a variety of mechanisms including defects in multimer assembly or an intrinsically increased sensitivity to cleavage by ADAMTS-13. The majority of pathogenic mutations are located in exon 28, predominantly affecting the A2 domain. Missense mutations have also been reported in the D3 and A1 domains (exons 22 and 25-28). Less common missense mutations include those responsible for dimerization (CK domain, exon 52) and multimerization defects (D2 domain, exons 11-17). Our objective was to determine the genomic distribution of mutations underlying type 2A VWD in patients referred to a USA-based clinical reference laboratory. Methods In a clinical reference laboratory, BloodCenter of Wisconsin, VWF gene sequence analysis results from January 2009 to July 2018 were reviewed. Molecular testing was performed by polymerase chain reaction (PCR) amplification and bi-directional Sanger sequencing or capture-based enrichment and next-generation sequencing (NGS). Complete coding region and splice junction of VWF gene was compared to the reference sequence (NM_00552.3). Variant interpretation was performed according to American College of Medical Genetics (ACMG) standards and guidelines by utilizing genome databases, population frequency data, variant specific literature search and predictive computational tools. Variant interpretation was reviewed by a team comprising of genetic counselors, molecular experts and experts in coagulation disorders correlating these findings with the patient's clinical phenotype provided. Results Pathogenic variants were identified in 46 cases received. Of these, 56.5 % (26 of 46) were in exon 28 of the VWF gene.(Figure 1) 21.7% of mutations (10 out of 46) were identified in exon 26, 4.3% were identified in exon 25 (2 of 46) and 6.5% (3 out of 46) were identified in exon 52 of VWF gene. The remaining 10.7% were identified in exons 6,11,12,22 and 27 (1 each). The distribution of these mutations according to VWF domains was also studied. 39% of these mutations were found in the A2 domain, 30.4% were identified in the D3 domain, and 13% were found in the A1 domain. A small number of these mutations were identified in the D1 domain (1/46), D2 domain (2/46) and CK domain (2/46). Phenotypic information was available for most patients evaluated. Conclusions In a predominantly Caucasian population with others of mixed ethnicities, the distribution of VWD type 2A mutations was studied in patients evaluated in a clinical reference laboratory. Most mutations were identified in VWF Exon 28 and in the A2 domain. Next generation sequencing platform enables sequencing of large amounts of DNA in parallel thereby allowing all VWF exons to be evaluated at one time. However, in smaller laboratories, without access to a NGS platform, initial evaluation of the VWF Exon 28 for evaluation of VWD type 2A variants is a cost effective measure. Subsequently, sequencing of other less implicated exons may be performed utilizing a reflexive algorithm. Genetic testing can be very useful in diagnosis of type 2 variants, where mutations in specific regions correspond to the defect in VWF function. Disclosures Friedman: Shire: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Description: The genetics of blood coagulation has been an ongoing area of research; and with the advent of next generation sequencing panels, there is a significant increase in the number of variants identified in coagulation factor genes. Several published reports and online databases document the variants observed in patients with bleeding disorders; however, the clinical interpretation of these variants is not always straight-forward. To enable gene-specific variant interpretation in coagulation factor deficiency disorders, the National Institutes of Health (NIH)-funded effort, Clinical Genome Resource (ClinGen), has developed the Coagulation Factor Deficiency Variant Curation Expert Panel (CFD-VCEP). The CFD-VCEP is comprised of expert clinicians, genetic counselors, clinical laboratory diagnosticians and researchers working toward the goal of developing and implementing standardized protocols for sequence variant interpretation for coagulation factor genes. The CFD-VCEP adapts the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for precise and consistent variant classification to genes involved in blood coagulation deficiencies. These guidelines recommend the use of 28 criteria codes based on the evidence category and the strength of the evidence (see Figure below). The first two genes under the purview of CFD-VCEP are F8 (OMIM: 300841) and F9 (OMIM: 300746). Pathogenic variants in the F8 and F9 genes resulting in the loss of protein function cause Hemophilia A and B, respectively. Owing to the similarity between these two genes with respect to their role in the coagulation cascade as well as the resulting phenotype, specification of variant curation guidelines for both genes has been undertaken simultaneously. With the completion of guideline specification for F8 and F9, the CFD-VCEP will subsequently continue this effort for other coagulation factor genes, while also curating F8 and F9 variants reported in ClinVar and other variant databases. Modifying the ACMG/AMP guidelines involves gene- and disease-informed specifications of the recommended criteria codes. This includes identifying which codes are applicable and which are not, defining gene- and disease-specific cut-offs such as for population frequency, and making code strength adjustments when appropriate. The specified guidelines are further refined based on their performance on a set of pilot variants (n = 30) for each gene compared to existing assertions of variant classification in ClinVar and by diagnostic laboratories represented in the CFD-VCEP. F8 and F9 variants classified by the CFD-VCEP will be submitted to ClinVar at the 3-star review status, with the tag of "FDA-recognized database", and the CFD-VCEP plans to begin this process by the second quarter of 2020. The considerations by the CFD-VCEP in the guideline-specification process and results from the pilot analysis will be discussed. This effort will lead to the standardized use of evidence criteria for the evaluation of variants in F8 and F9, which will reduce the number of variants of uncertain significance and those of conflicting interpretations, making genetic testing results more informative for providers and patients. The CFD-VCEP also encourages sharing de-identified data on variants among laboratories, which enables accurate and consistent curations. Figure Disclosures Lee: UNC Hemophilia Treatment Center: Employment. Carcao:Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees; Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire/Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Research Funding; LFB: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bioverativ/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kemball-Cook:European Association for Haemophilia and Allied Disorders: Other: Freelance . Leebeek:CSL Behring: Research Funding; uniQure BV: Consultancy, Research Funding; Baxalta/Shire: Research Funding. Miller:Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention: Consultancy.