Genetics, Pathophysiology, and Treatment of Recessive Autoinflammatory Diseases

National Human Genome Research Institute

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Abstract

During the current reporting period we have focused on three areas: 1) Discovery of a patient with homozygous SHARPIN deficiency With HOIL-1 and HOIP, SHARPIN encodes one of the three major structural proteins in the linear ubiquitin assembly complex (LUBAC), which is essential for post-translational modification of proteins with linearly linked ubiquitin chains. About 10 years ago Jean-Laurent Casanovas group described disorders caused by mutations in HOIL-1 and HOIP that present with both autoinflammation and immunodeficiency. Functional data from patients suggested that hyperresponsiveness of monocytes to proinflammatory cytokines explained the autoinflammation, and that impaired canonical NF-kappaB activation in B cells accounted for the immunodeficiency. Since the initial description of HOIL-1 deficiency a decade ago, no human patients had been described with SHARPIN deficiency. A spontaneous mutation in the murine homolog had been described in the cpdm mouse line. Homozygotes exhibit a chronic proliferative dermatitis and multi-organ autoinflammatory disease that is driven by TNF-dependent cell death. But the human version of homozygous SHARPIN deficiency remained elusive. In a previous reporting period, we identified a single patient, the son of an uncle-niece marriage from Bangalore, India, with a homozygous frame-shift mutation in SHARPIN that is within a few residues of the corresponding cpdm frame shift in murine Sharpin. We propose the term sharpenia to denote the clinical disorder caused by SHARPIN deficiency. In contrast to cpdm mice, the patient has no cutaneous disease, although he does exhibit several other inflammatory manifestations, including parotitis, colitis, and arthritis. In contrast to the patients described with HOIL-1 or HOIP mutations, the SHARPIN-deficient patient manifests relatively subtle immunodeficiency disease. To understand the differences, we conducted a series of mechanistic studies at the NIH Clinical Center that have included samples from the Indian sharpenia patient as well as from patients with homozygous loss-of-function mutations in HOIL-1, HOIP, and OTULIN (which encodes an enzyme that removes linear ubiquitin chains), and from patients with CRIA syndrome (caused by dominantly inherited cleavage resistant RIPK1). Our data demonstrated a common mechanism that transcends all five diseases: increased TNF-dependent cell death that drives autoinflammation. Based on this discovery, we initiated an empiric trial of TNF inhibitors in our sharpenia patient, and we documented a prolonged dramatic improvement in his arthritis and colitis. We also sought to characterize the immunodeficiency of sharpenia. Fortunately for these studies, on a checkup visit to the NIH Clinical Center our Indian patient presented with otitis media. Ear-nose-and-throat consultants recommended an adenoidectomy, which afforded the opportunity for histologic and high-dimensional flow cytometric analysis of secondary lymphoid tissue. These studies demonstrated a marked impairment of germinal center B cell development and reduction in numbers of germinal center follicular helper T cells. Subsequent experiments showed that EBV B cell lines from the sharpenia patient exhibited diminished NF-kappaB activation induced by CD40L, but increased CD40L-driven cell death in primary B cells. We also observed increased caspase-3 cleavage around the lymphoid follicles from the sharpenia patient and around the lymphoid follicles of an axillary lymph node of a HOIP deficient patient. Hence, we found that for two members of the TNF superfamily (TNF and CD40L), LUBAC deficiency is associated with decreased canonical NF-kappaB signaling and increased apoptosis, which cause both humoral immunodeficiency and autoinflammation. A manuscript describing these findings in detail and proposing the overarching concept of inborn errors of cell death has been submitted and is currently undergoing revisions. 2) Clinical and mechanistic studies of the deficiency of adenosine deaminase 2 (DADA2) Our laboratory participated in a large collaborative transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) in 33 patients with DADA2. Transcriptomic analysis of PBMCs from DADA2 patients with the vasculitis phenotype or pure red cell aplasia phenotype exhibited similar upregulation of TNF, type I interferon, and type II interferon signaling pathways compared with healthy controls. These pathways were also activated in 3 asymptomatic individuals with DADA2. Analysis of ADA2 variants, including 7 novel variants, showed different mechanisms of functional disruption including (1) unstable transcript leading to RNA degradation; (2) impairment of ADA2 secretion because of retention in the endoplasmic reticulum; (3) normal expression and secretion of ADA2 that lacks enzymatic function; and (4) disruption of the N-terminal signal peptide leading to cytoplasmic localization of unglycosylated protein. Based in part on clinical experiences with the large cohort of patients followed at the NIH Clinical Center, our group helped lead the development of an International Consensus Statement on the evaluation and management of DADA2. This included the definition of DADA2 disease phenotypes, statements with regard to the relative merits of genetic and biochemical testing, statements pertaining to screening for DADA2, and statements related to the clinical evaluation and management of DADA2. 3) Collaborative studies on familial Mediterranean fever By providing various knock out and knock in mice, we have contributed to studies of the role of neuroimmune and endocrine factors in familial Mediterranean fever (FMF). Collaborators at the Feinstein Institute for Medical Research demonstrated that galantamine, an acetylcholinesterase inhibitor, attenuated autoinflammation in FMF knock in mice. Collaborators in Lyon, France, identified sex hormone catabolites as specific activators of pyrin, the protein mutated in FMF.