Abstract: Introduction: Low von Willebrand factor (VWF) activity is prevalent in adolescents with heavy menstrual bleeding (HMB). There is a need to better genetically characterize these patients and improve our understanding of the pathophysiology of their bleeding risk. Methods: One of the main objectives of this multi-center, single arm, observational cohort study was to genotype adolescent females with HMB and low VWF (≥ 30 and ≤ 50 IU/dL) by means of whole exome sequencing (WES), to identify variants throughout the exome that may modulate risk for bleeding. Post-menarchal females & lt; 21 years with HMB (defined as PBAC score & gt;100) and low VWF were eligible for the study. All patients were enrolled by participating centers where blood samples were collected. Exome data for 86 cases and 900 unrelated pediatric ( & lt;21 years) controls taken from a genetic study on Chiari 1 Malformation (CM1) were aligned and sorted, and variants called using the Sentieon software package. Annotation including allele frequencies, function, amino acid change and Clinvar rating among others was obtained using ANNOVAR. Variants were retained if there was a genotype call rate of & gt;0.8, GQ & gt;20, AB between 0.3-0.7 and min DP & gt;8. Case/control analyses included common and rare SNP associations, gene burden analysis of both rare nonsynonymous variants and ClinVar 'pathogenic' variants, and gene-set burden analyses. Variants were considered rare if they had a minor allele frequency of & lt;1% in the Genome Aggregation Database (gnomAD). Count differences between cases/controls were determined by Fisher's Exact test. Results: Of the 113 subjects enrolled, 86 had sufficient blood samples collected for WES. The median age was 16.2 years (range: 11.5-19.6). 36% of cases showed variants in VWF vs. 26% of controls (p=0.14). After multiple test correction, WES revealed a significant common variant association in FERMT2 with an LD block with a frequency of 24% in cases and 6% in controls (p=7.5x10-7). Rare variant analysis showed a significant association with an intronic variant in ABCA13 (p=1.6x10-7). Among the gene burden analysis using rare nonsynonymous variants, 2 genes of interest passed multiple test correction: IL12B and DTNBP1. When using ClinVar 'pathogenic' variants as the input for the gene burden analysis, 4 genes of interest passed multiple test correction: HBM, MYLK, RUNX1 and CD36. Gene-set burden analysis revealed 5 significant pathways of interest, including platelet degranulation, platelet alpha granule lumen and erythrocyte differentiation. We then focused a subset of known risk genes and compared the number of missense, nonsense and ClinVar 'pathogenic' variants between cases and controls. GP6, and MTHFR had significantly more missense variants in controls vs. cases (p=0.003 and 0.01, respectively), and F13B approached significance, with more missense variants in controls than cases (p=0.07). Conclusion: We found VWF variants in 36% of subjects, in accordance with previous reports. We found novel SNP associations with variants in FERMT2 and ABCA13.FERMT2 encodes for the integrin, Kindlin 2, which has been shown to be critical for supporting vascular integrity. Several other relevant genes passed multiple test correction in gene burden analyses, including genes known to cause Hermansky-Pudlak syndrome (DTNBP1), familial platelet disorder (RUNX1), platelet glycoprotein IV deficiency (CD36) and aortic aneurysm (MYLK). This association with platelet disorders was strengthened by our gene-set burden results, which implicate several platelet-related pathways. These data suggest that while a subset of HMB patients may have their bleeding explained by variants in VWF, there is a role for other hemostasis, platelet biology and vascular integrity related gene variants which can contribute to the variation in bleeding severity in adolescents with low-VWF related HMB. Study supported by an investigator-initiated research grant from Shire US Inc., now part of Takeda Disclosures O'Brien: Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mullins:Takeda, Bayer: Other: Advisory Board. Sidonio:Takeda: Research Funding. Ragni:Sangamo: Consultancy, Research Funding; Takeda: Research Funding; Bioverativ: Consultancy, Research Funding; Spark: Consultancy, Research Funding; BioMarin: Consultancy, Research Funding; Alnylam/Sanofi, ATHN, BioMarin, Bioverativ, Sangamo, Spark: Research Funding; Alnylam/Sanofi, BioMarin, Bioverativ, Spark: Consultancy; Alnylam Pharmaceuticals Inc., Baxalta/Takeda, BioMarin, Bioverativ, and Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; American Thrombosis Hemostasis Network: Other: Committee work; Baxalta/Takeda, CSL Behring, Genentech, a member of the Roche Group, OPKO Biologics, and Vascular Medicine Institute: Research Funding. Kulkarni:Sanofi/ Bioverativ, Bayer, Biomarin, Shire/Takeda, Novo Nordisk, Freeline: Other: clinical trial research grants ; Bioverativ/Sanofi, BPL, Genentech, Kedrion, Novo Nordisk, Octapharma, Pfizer, Takeda, Catalyst Bioscience Bayer: Membership on an entity's Board of Directors or advisory committees. Srivaths:Shire US Inc., now part of Takeda: Research Funding.

Abstract: Adolescents with low von Willebrand factor (VWF) levels and heavy menstrual bleeding (HMB) experience significant morbidity. There is a need to better characterize these patients genetically and improve our understanding of the pathophysiology of bleeding. We performed whole-exome sequencing on 86 postmenarchal patients diagnosed with low VWF levels (30-50 IU/dL) and HMB and compared them with 660 in-house controls. We compared the number of rare stop-gain/stop-loss and rare ClinVar “pathogenic” variants between cases and controls, as well as performed gene burden and gene-set burden analyses. We found an enrichment in cases of rare stop-gain/stop-loss variants in genes involved in bleeding disorders and an enrichment of rare ClinVar “pathogenic” variants in genes involved in anemias. The 2 most significant genes in the gene burden analysis, CFB and DNASE2, are associated with atypical hemolytic uremia and severe anemia, respectively. VWF also surpassed exome-wide significance in the gene burden analysis (P = 7.31 × 10−6). Gene-set burden analysis revealed an enrichment of rare nonsynonymous variants in cases in several hematologically relevant pathways. Further, common variants in FERMT2, a gene involved in the regulation of hemostasis and angiogenesis, surpassed genome-wide significance. We demonstrate that adolescents with HMB and low VWF have an excess of rare nonsynonymous and pathogenic variants in genes involved in bleeding disorders and anemia. Variants of variable penetrance in these genes may contribute to the spectrum of phenotypes observed in patients with HMB and could partially explain the bleeding phenotype. By identifying patients with HMB who possess these variants, we may be able to improve risk stratification and patient outcomes.

Abstract: Approximately 35% of patients with type 1 von Willebrand disease (VWD) do not have a known pathogenic variant in the von Willebrand factor (VWF) gene. We aimed to understand the impact of VWF coding variants on VWD risk and VWF antigen (VWF:Ag) levels, studying 527 patients with low VWF and VWD and 210 healthy controls. VWF sequencing was performed and VWF:Ag levels assayed. A combined annotation-dependent depletion (CADD) score & gt;20 was used as a predicted pathogenicity measure. The number of rare nonsynonymous VWF variants significantly predicted VWF:Ag levels (P = 1.62 × 10−21). There was an association between average number of rare nonsynonymous VWF variants with VWD type 1 (P = 2.4 × 10−13) and low VWF (P = 1.6 × 10−27) compared with healthy subjects: type 1 subjects possessed on average & gt;2 times as many rare variants as those with low VWF and 8 times as many as healthy subjects. The number of rare nonsynonymous variants significantly predicts VWF:Ag levels even after controlling for presence of a variant with a CADD score & gt;20 or a known pathogenic variant in VWF (P = 2.7 × 10−14). The number of rare nonsynonymous variants in VWF as well as the presence of a variant with CADD & gt;20 are both significantly associated with VWF levels. The association with rare nonsynonymous variants holds even when controlling for known pathogenic variants, suggesting that additional variants, in VWF or elsewhere, are associated with VWF:Ag levels. Patients with higher VWF:Ag levels with fewer rare nonsynonymous VWF gene variants could benefit from next-generation sequencing to find the cause of their bleeding.

Abstract: Background: The known driver mutations in PMF (including JAK2, CALR, and MPL) do not explain the highly inflammatory phenotype associated with this disease. Moreover, JAK2 inhibitors provide symptomatic relief in PMF, but they are not curative, indicating that our understanding of the molecular pathology of MPNs is incomplete. High-resolution insights into the mutational landscape of MPNs may inform new diagnostic and therapeutic approaches. Methods: We performed 60x whole-genome sequencing (WGS) on CD34+ hematopoietic stem/progenitor cells and matched in vitro-expanded CD3+ lymphocytes from 10 patients (pts) with PMF, of whom 5 had JAK2 V617F, 2 had MPL W515L, and 3 had CALR (del52 or ins5) mutations. Paired-end WGS was performed using BGISEQ-500 platform. Clean reads with Q20 and Q30 at 96.4% and 87.1%, respectively, were aligned to the human GRCh37 reference genome. 99.9% reads mapped successfully and 90.6% mapped uniquely. Mean sequencing depth for CD3+ and CD34+ cells were 72x and 83x, respectively (Fig A). Bioinformatic pipeline strategy is summarized on Fig B. We also examined complement activity in sera from 10 PMF pts using CH50 (a screening test for total complement activation) and C3 and C4 component activity. We further evaluated the effect of complement-neutralizing anti-C3 and anti-C5 (C3/C5 NeuAbs) on JAK/STAT and NF-κB signaling in mononuclear cells (MNCs) isolated from peripheral blood of these PMF pts. Results: ~3.4e6 SNPs were identified (n=10): 99.9% were represented in dbSNP, 98.1% were annotated in GnomAD, and 4,587 were novel. Among ~8.6e5 InDels, 91.9% were represented in dbSNP, 53.8% in GnomAD, and ~6.9e4, were novel. We identified 3,540 copy-number variations (CNVs) and 3,365 SVs. Rare non-synonymous variants (RNSV) were defined as SNVs with a maximum minor allele frequency & lt;0.01 in any GnomAD population. RNSVs were further filtered based on clinical interpretation of their genomic variation in ClinVar. 78 unique RNSVs were identified (Fig C), after exclusion of RNSVs with benign, likely benign, or undetermined status. Known PMF drivers (JAK2 V617F, CALR, and MPL W515L) were present in both CD34+ and CD3+ cells, except in 2 samples JAK2 V617F was present only in CD34+ cells. This suggested WGS-detectable driver clonality in most but not all analyzed cases. All mutations except MPL were heterozygous. We further found known RNSVs in TP53, U2AF1, TCF12, as well as previously unreported in PMF pathogenic mutations in CD247 and OTUD6B. Among 11 RNSVs with conflicting classification in ClinVar, we observed mutations in BRCA2, TTN, APOB, ATM and CDH1 (previously linked to MPNs). Within this group of variants, we also detected a p.G119R mutation in Complement Factor I (CFI, rs141853578) in 2 samples. This RNSV was not previously reported in PMF. CFI G119R was detected in 1 sample with JAK2 and 1 with CALR driver mutation, in both CD34+ and CD3+cells as confirmed by Sanger sequencing (Fig D). CFI encodes a serine proteinase that regulates the complement pathway, and its deficiency is associated with severe inflammatory pathology. In the sera of different cohort of 10 pts with PMF, we observed an increase in CH50 levels (n=3) and depletion of C3 (n=3; Fig E), independent of the presence of specific driver mutations or treatment with ruxolitinib (RUXO) or hydroxyurea, suggesting that complement overactivation may contribute to the pathology of PMF in some pts. We further incubated PMF or healthy PBMCs in matching plasmas in the presence of C3/C5 NeuAbs either alone or in combination with RUXO. In PBMCs from healthy controls, STAT3 pTyr705 decreased upon treatment with RUXO alone or with C3/C5 NeuAbs, and NF-κB pSer536 decreased for all treatment conditions (Fig F). In contrast, in PMF, STAT3 pTyr705 did not consistently decrease in response to RUXO either alone or with C3/C5 NeuAbs. Notably, NF-κB pSer536 was unaffected by RUXO alone, and only treatment with RUXO + C3/C5 NeuAbs induced a decrease in NF-κB pSer536 (Fig F). Conclusions: Using WGS, we discovered an inactivating RNSV in CFI G119R in 20% of pts with PMF. In a separate cohort of PMF patients we detected elevated serum complement activation. Furthermore, PMF-specific dysregulation of STAT3 and NF-κB activation could be modulated by exposure to C3/C5 NeuAbs. This is a first, to our knowledge, report linking the overactivation of the complement cascade to inflammation-related pathogenesis of PMF. Figure 1 Figure 1. Disclosures Hobbs: Novartis: Consultancy; Bayer: Research Funding; Celgene/Bristol Myers Squibb: Consultancy; AbbVie.: Consultancy; Constellation Pharmaceuticals: Consultancy, Research Funding; Incyte Corporation: Research Funding; Merck: Research Funding. Oh: Abbvie: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Celgene Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Membership on an entity's Board of Directors or advisory committees; PharamaEssentia: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees. Di Paola: CSL Behring: Consultancy, Honoraria.