Genetic Analysis of Complex Inflammatory Disorders

National Human Genome Research Institute

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Abstract

During the current reporting period, most of our efforts were focused on using a novel genomic strategy to account for adult-onset undiagnosed cases of recurrent fever and inflammation in our NIH clinic. This effort was led by David Beck, a Human Genetics Clinical Scholar in the laboratory. The project was a particular challenge because parental DNA is often unavailable for trio analysis in these patients. Rather than adopting a conventional approach, that of grouping patients with similar phenotypes together and then attempting to find a genetic variant in common among these subjects and absent in controls, David adopted a genotype first approach. Based on the fact that our laboratory had already discovered monogenic autoinflammatory diseases caused by mutations in 2 genes encoding deubiquitylases (TNFAIP3 and OTULIN), he reasoned that variants in other genes encoding regulators of ubiquitylation might also cause autoinflammatory diseases. He therefore drew up a list of 841 human genes regulating ubiquitylation and used the list to interrogate 1477 undiagnosed exomes from our own database and 1083 exomes from the Undiagnosed Diseases Program (UDP). As filtering criteria, David selected genes intolerant to haploinsufficiency (pLI > 0.9) and variants absent in gnomAD but shared among cases. From this analysis, he identified 3 middle-aged men who appeared to be heterozygous for a mutation in an important ubiquitin-regulatory gene encoded on the X-chromosome. Barring aneuploidy (which we ruled out), this seemed highly unlikely (most men have only a single X-chromosome), and standard filtering software are programmed to exclude heterozygous reads for X-chromosome genes in men as likely sequencing errors. However, David correctly reasoned that somatic mosaicism might be the explanation, and further testing showed that these 3 patients were hemizygous for myeloid-restricted somatic mutations in this gene. Erythroid and myeloid precursors in the bone marrow of all 3 patients exhibited vacuoles. We were quickly able to identify an additional 22 subjects with overlapping clinical phenotypes from our own cohort and through colleagues in the NIAMS. All 25 of these men had somatic mutations affecting methionine-41 in UBA1, the major E1 enzyme that initiates ubiquitylation, and thus we denoted their illness VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. All 25 had developed an often-fatal, treatment refractory inflammatory syndrome in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweets syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome MDS or multiple myeloma) or both. Mutations were found in more than half of the hematopoietic stem cells and peripheral-blood myeloid cells, but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic p.Met41-initiated isoform of UBA1 and in expression of a catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knock-out of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation. Subsequent to the publication of this work in the New England Journal of Medicine (online in October timed to coincide with the opening plenary session of the American Society of Human Genetics meeting last year, in print on New Years Eve), we are now aware of over 100 VEXAS cases in the U.S. and many more worldwide. Based on a preliminary scan of a large exome database, the population frequency of VEXAS syndrome is approximately 1:30,000, suggesting a total of 10,000 cases in the U.S. Based on anecdotal cases in which patients thought to have another condition actually were later found to have VEXAS syndrome, hematopoietic stem cell transplantation (HSCT) is likely to be effective, perhaps even curative. More importantly, VEXAS has established a paradigm for investigating other genetically complex diseases of adulthood, both for somatic mutations in genes expressed in the peripheral blood and for germline mutations. This might involve substituting some other gene list for the list of ubiquitylation-related genes, or to query additional large unrelated cohorts with relatively ill-defined (or broadly defined) inflammatory phenotypes, or to modify the filtering criteria. Possibly, such a strategy will uncover additional somatic or germline mutations that refine or cut across clinical diagnoses. Overall, there are three major conclusions that can be drawn from this study. First, using a genotype-first strategy, one can define new illnesses based on genetic variants shared among patients carrying distinct clinical diagnoses, and this may give rise to a new molecular taxonomy of rheumatic disease. Seconod, somatic mutations for genes encoded on the X-chromosome are probably under-recognized. And third, somatic mutation may account for a significant fraction of adult-onset inflammatory/rheumatic disease. Altogether, this project resulted in 10 of the publications listed for this reporting period.