Genetic Analysis of Complex Inflammatory Disorders

National Human Genome Research Institute

Investigators

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Abstract

African-American Scleroderma We participated in a collaborative project with Pravitt Gourh, a former fellow and now Assistant Clinical Investigator in the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), to study the role of rare and low frequency genetic variants in the pathogenesis of scleroderma in African Americans. Exome and targeted sequencing were performed in discovery and replication cohorts comprising 969 African-American patients and 771 controls. We assembled a list of 32 genes previously associated with scleroderma in genome-wide association studies (GWAS) and used this list to interrogate the sequence from 379 African-American scleroderma patients and 411 controls in the discovery cohort and 590 African-American scleroderma patients and 360 controls in the replication cohort, using a gene-based sequence kernel association test (SKAT). Only one of the 32 candidate genes, NOTCH4, was associated with scleroderma at an exome-wide significance (P=1.6x10-7) and in patients with severe vascular disease (P=3.5x10-7). The risk haplotype defined by the missense (c.2824C>T) and promoter (c.-117G>A) variants was enriched in African Americans with scleroderma vs. controls, and increased risk of the combination of severe Raynauds, renal crisis, and pulmonary arterial hypertension (OR=10.6, 95% CI 2-56). The c.-117A allele, residing in the glucocorticoid receptor binding site, was associated with increased NOTCH4 transcripts and protein expression. The c.2824 allele upregulated NOTCH4 signaling in lymphoblastoid cell lines. Scleroderma skin single-cell RNA sequencing identified NOTCH4 expression principally in endothelial cells, with dysregulated angiogenesis and endothelial-to-mesenchymal transition. NOTCH4 stimulation in an endothelial cell line and in c.-117A scleroderma primary endothelial cells attenuated tube formation. Inhibiting NOTCH4 rescued normal tube formation. In this study we concluded that NOTCH4 variants are associated with scleroderma pathogenesis and vasculopathy, partly explaining the increased prevalence of scleroderma in African Americans. Blocking NOTCH4 signaling restored angiogenesis. A manuscript detailing these findings has been submitted. VEXAS Syndrome We have participated in 3 collaborative projects focused on VEXAS syndrome. The first was published in the Journal of the American Medical Association in January 2023, and involved a retrospective observational study evaluating UBA1 variants in exome data from 163,096 participants from the Geisinger MyCode Community Health Initiative. The participants were 94% White, 61% women, and had a mean age of 52.8 years. Eleven individuals harbored likely somatic variants at known pathogenic UBA1 positions (variant allele fractions from 4% to 79%) with 11/11 having clinical manifestations consistent with VEXAS syndrome (9 M, 2 F). 5/11 did not meet criteria for rheumatologic and/or hematologic diagnoses previously associated with VEXAS syndrome. All 11 mutation-positive individuals had anemia (mean Hgb 7.8 g/dL), 10/11 macrocytic, and 10/11 had thrombocytopenia. Among the 11 patients identified, there was a pathogenic variant in 1 male participant prior to onset of VEXAS-related signs or symptoms and 2 female participants had disease with heterozygous variants. A previously unreported UBA1 variant (p.Ser621Cys) was found in a male patient carrying the diagnosis of granulomatosis with polyangiitis, with in vitro data supporting a catalytic defect and pathogenicity. Altogether, disease-causing UBA1 variants were found in 1 in 13,591 unrelated individuals, 1 in 4269 men over the age of 50 years, and 1 in 26,238 women older than 50 years. Two other collaborative papers examined the pathogenesis of VEXAS syndrome. The first, published in Blood, examined the spectrum of clonal hematopoiesis in 80 patients (64 from the NIH, 16 from the Mayo Clinic) with VEXAS syndrome. 74/77 patients had UBA1 mutations at p.Met41. Typical clonal hematopoiesis mutations cooccurred with UBA1 mutations in 60% of patients, mostly in DNMT3A and TET2, and were not associated with inflammatory or hematologic manifestations. By single-cell sequencing, clonal hematopoiesis could either precede or follow the development of UBA1 mutations. DNMT3A-positive clones had a variant allele fraction of 25%, while TET2-positive clones had a 1% variant allele fraction. Overall patient survival was 60% at 10 years, with transfusion-dependent anemia, moderate thrombocytopenia, and typical clonal hematopoiesis each correlating with poor outcome. The second collaborative project focusing on VEXAS pathogenesis was recently published in Cell Reports Medicine. In a study of single-cell transcriptomics in the bone marrow of VEXAS patients, we found that myeloid lineage bias and inflammatory gene activation occur early in hematopoietic stem cells and appear intrinsic to UBA1 mutant cells. There was evidence for TNF and IFN-gamma-mediated cell-cell interactions among hematopoietic stem and progenitor cells, suggesting the possibility of autocrine feedback loops. Dysregulation in protein degradation was associated with higher stress response in VEXAS stem and progenitor cells, which positively correlated with inflammation. Apoptosis of mutant UBA1 lymphoid progenitors caused impaired lymphopoiesis in VEXAS. While B-cell numbers were decreased, the B cell receptor repertoires were preserved. In contrast, T-cell receptor usage was restricted and there was increased cytotoxicity and IFN-gamma signaling in T cells.