Rare Variant Genetic Association Study for Transplant-Associated Thrombotic Microangiopathy (TA-TMA) Via Whole Exome Sequencing

Introduction: Transplant-associated thrombotic microangiopathy (TA-TMA) is an increasingly recognized hematologic complication after allogeneic hematopoietic cell transplantation (HCT). While few studies have reported germline association with rare variants in complement genes using targeted next generation sequencing (NGS) method, they were limited by small sample size (≤40 TMA cases) and lack of analysis of non-complement genes (PMIDs 26603840, 32131130). In the present study, we employed whole exome sequencing (WES) to assess rare variant contribution to the development of TMA in a hypothesis-driven pathway-specific approach. Methods: In the current case-control genetic association study conducted at Fred Hutchinson Cancer Research Center, we selected 100 patients with a diagnosis of TMA and pre-transplant DNA samples (case definition described previously in PMID 30940363, 33836868). We then performed incidence density sampling to randomly select 100 non-TMA controls after allogeneic HCT matching by age, sex, race, and year of HCT. WES (germline variant detection 40x) was conducted using Illumina NovaSeq. Sequence reads were mapped to hg38 reference genome followed by deduplication and base quality score recalibration. Joint-genotyping was performed to call single nucleotide polymorphism (SNPs) and insertion/deletion (indels) using the GATK v3.3 and Atlas2. Variants were filtered during quality control (QC) and variant quality score recalibration (VQSR) and annotated using ANNOVAR and Ensembl VEP. To optimize signal detection by reducing neutral background variation, we defined qualifying variants as those meeting all 3 criteria: 1) novel or rare variants with a minor allele frequency (MAF) <1% in the reference database gnomAD; 2) functional variants with missense, frameshift, indel, splice region/acceptor/donor, start/stop gained/lost, coding sequence, and protein altering in VEP; and 3) missense variants previously reported to be likely pathogenic from the ClinVar database or predicted to be deleterious from 4/6 in-silico prediction tools (SIFT, Polyphen-2, MutationTaster, MutationAssessor, FATHMM, and FATHMM-MKL) (Figure 1). We then focused on the exome profiles of 5 a priori selected genetic pathways: complement regulation (17 genes), VWF and coagulation (7 genes), VWF clearance (10 genes), ADAMTS13 mimics or interacting proteins (10 genes), and angiopoietin family and endothelial activation (7 genes). Pathway-based and gene-based collapsing association tests were performed using the Optimized Sequence Kernel Association (SKAT-O) test as an optimal test combining burden test and SKAT. Results: After joint variant calling, 91 TMA cases and 93 non-TMA controls passed all QC filters (Table 1). Among 1,485 variants detected in the 5 pathways after QC, 60 variants (73 total mutations) were considered as qualifying variants with MAF <1%, functional coding, and in-silico pathogenic prediction (Figure 1). From pathway-based analysis, a significant association was observed in the VWF clearance pathway (p=0.041) but not in the complement regulation pathway (p=0.308) or the other 3 pathways (Table 2). From gene-based analysis, the significant association in the VWF clearance pathway appeared to be driven by rare variants within the LRP1 gene (Figure 2), which encodes a member of the low-density lipoprotein receptor family of proteins that contributes to the clearance of VWF (PMID 22234691). Sensitivity analyses performed including all rare variants without in-silico pathogenicity prediction resulted in similar findings. Conclusion: Contrary to the initial hypothesis, we did not observe pathogenic germline rare variants in the complement regulation pathway in patients with TA-TMA. Instead, we found a significant association in the VWF clearance pathway, particularly that of the LRP1 gene. In recent years, researchers have shown that VWF can bind to and activate complement proteins. Impaired VWF clearance could lead to the higher predisposition for complement activation observed in patients with TA-TMA. Future functional studies are needed to determine the impact of VWF clearance on the pathogenesis of the disease. Figure 1 Figure 1. Disclosures Sartain: Alexon Pharamaceuticals: Membership on an entity's Board of Directors or advisory committees. Lee: Incyte: Research Funding; Janssen: Other; Takeda: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Syndax: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding.

Severe Hemolysis in a COVID-19 Patient with Paroxysmal Nocturnal Hemoglobinuria

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), has been demonstrated to be able to activate complement, making patients with deficiency in negative complement regulation, such as paroxysmal nocturnal hemoglobinuria (PNH), particularly vulnerable to complement-mediated cell damage. We report a case of a patient who presented with fatigue, facial swelling, and upper respiratory infection (URI) symptoms and was found to have COVID-19 with laboratory tests showing severe hemolysis and pancytopenia secondary to PNH.

Potentiation of complement regulator factor H protects human endothelial cells from complement attack in aHUS sera

Mutations in the gene encoding for complement regulator factor H (FH) severely disrupt its normal function to protect human cells from unwanted complement activation, resulting in diseases such as atypical hemolytic uremic syndrome (aHUS). aHUS presents with severe hemolytic anemia, thrombocytopenia, and renal disease, leading to end-stage renal failure. Treatment of severe complement-mediated disease, such as aHUS, by inhibiting the terminal complement pathway, has proven to be successful but at the same time fails to preserve the protective role of complement against pathogens. To improve complement regulation on human cells without interfering with antimicrobial activity, we identified an anti-FH monoclonal antibody (mAb) that induced increased FH-mediated protection of primary human endothelial cells from complement, while preserving the complement-mediated killing of bacteria. Moreover, this FH-activating mAb restored complement regulation in sera from aHUS patients carrying various heterozygous mutations in FH known to impair FH function and dysregulate complement activation. Our data suggest that FH normally circulates in a less active conformation and can become more active, allowing enhanced complement regulation on human cells. Antibody-mediated potentiation of FH may serve as a highly effective approach to inhibit unwanted complement activation on human cells in a wide range of hematological diseases while preserving the protective role of complement against pathogens.