Multiple myeloma arises when malignant plasma cells invade and form multiple tumors in the bone marrow. High levels of heparanase (HPSE) correlate with poor prognosis in myeloma patients. A likely target of the enzyme is the heparan sulfate (HS) proteoglycan syndecan-1 (Sdc1, CD138), which is highly expressed on myeloma cells and contributes to poor prognosis in this disease. We find that HPSE promotes an invasive phenotype mediated by the very late antigen-4 (VLA-4, or α4β1 integrin) in myeloma cells plated on either fibronectin (FN) or vascular endothelial cell adhesion molecule-1 (VCAM-1), ligands that are prevalent in the bone marrow. The phenotype depends on vascular endothelial cell growth factor receptor-2 (VEGFR2), which is aberrantly expressed in myeloma, and is characterized by a highly protrusive lamellipodium and cell invasion. HPSE-mediated trimming of the HS on Sdc1 and subsequent matrix metalloproteinase-9-mediated shedding of the syndecan exposes a juxtamembrane site in Sdc1 that binds VEGFR2 and VLA-4, thereby coupling VEGFR2 to the integrin. Shed Sdc1 can be mimicked by recombinant Sdc1 ectodomain or by a peptide based on its binding motif, which causes VLA-4 to re-orient from the lagging edge (uropod) to the leading edge of migrating cells, couple with and activate VEGFR2. Peptides (called 'synstatins') containing only the VLA-4 or VEGFR2 binding sites competitively inhibit invasion, as they block coupling of the receptors. This mechanism is also utilized by vascular endothelial cells, in which it is also activated by HPSE, during endothelial cell tube formation. Collectively, our findings reveal for the first time the mechanism through which HPSE modulates Sdc1 function to promote both tumor cell invasion and angiogenesis, thereby driving multiple myeloma progression. The inhibitory synstatins, or inhibitors of HPSE enzyme activity, are likely to show promise as therapeutics against myeloma extravasation and spread.

Atypical Hemolytic Uremic Syndrome (aHUS) is a rare disease of hemolysis, thrombocytopenia, and organ dysfunction (predominantly renal or CNS) that is often attributed to mutations in the alternate pathway of the complement system. To aid in the evaluation of patients with aHUS, a 15-gene next generation sequencing (NGS) panel was developed. Included in the panel are several genes within the highly homologous region of complement activation (RCA). Sixteen exon pairs across five genes in this region (CFH, CFHR1, CFHR3, CFHR4, and CFHR5) have greater than 95% sequence identity. This leads to difficulties in aligning corresponding NGS reads to the appropriate exons. Accordingly, reference databases for normal populations, as generated by whole genome or whole exome sequencing, are lacking in variant frequency data for these and many other highly-homologous genes, leaving an increased dependence on predictive tools in attempt to classify the pathogenicity of variants. A detail-oriented approach, including allele-specific PCR and Sanger sequencing of longer amplicons to confirm appropriate alignment of reads, allowed for interrogation of these difficult regions for which polymorphism frequency data is sparse. An example of a challenging NGS alignment is reads attributed to CFHR3 exon 5 or CFHR4 exon 9. The reference sequences for these exons differ by one base pair, which correspond to CFHR3 c.721 C and CFHR4 c.1415 T. Combining allele-specific PCR and Sanger sequencing uncovered a variant in CFHR3 (c.721C>T) which corresponds to the reference nucleotide for CFHR4. Allele-specific PCR involved designing primers in the introns of these genes which results in sequencing a region 770 bases long, or more than three times the length of a typical NGS read. Due to the length of the sequence identity, the NGS read corresponding to this variant always aligned bioinformatically to the incorrect gene (CFHR4) as the reference allele. The variant was analyzed with several algorithms including SIFT, Polyphen2, Condel, and Mutation Taster which predicted it was benign, but the accuracy of these tools is uncertain. This variant was not previously reported in either aHUS patients or the normal population; however, over a 16 month period, allele-specific PCR and Sanger sequencing for this variant of unknown significance was performed on all patient samples and was detected in 14% of the samples tested. In order to better categorize the pathogenicity of the variant, it was necessary to determine whether the subset of patient DNA tested was enriched for the variant because it was associated with aHUS, or whether the variant was present at such a high frequency in the normal population. A high-throughput melt-curve assay was developed. CFHR3 exon 5 and a portion of the adjacent introns was amplified by allele specific PCR and melting curve analysis was performed to determine the genotype using FRET probe technology on the LC480 instrument. DNA extracted from normal blood donor samples, including 96 African Americans, 74 Caucasians, and 112 Hispanics were screened. Overall, 31% of the population was heterozygous for the variant and 16% of the population was homozygous for the variant. The allele was most common in the African American population where 25% of the population was homozygous for the variant and least common in the Caucasian population where 68% of the population was wild-type. This data provides evidence that the variant is benign and not associated with an increased risk of aHUS. Focused large-gene panels, such as this one for aHUS, highlight the ability to meet challenges associated with NGS technology in regions of high sequence identity. We presented our approach for resolution of sequencing information to allow for appropriate classification of variant pathogenicity, resulting in decreased reporting of clinically insignificant results. Disclosures Friedman: Novo Nordisk: Consultancy; Alexion: Speakers Bureau; Instrumentation Laboratories: Consultancy; CSL Behring: Consultancy, Honoraria.

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.