During the current reporting period we focused on the following projects: 1) Familial Mediterranean fever (FMF) This project represents the convergence of two lines of investigation in our laboratory. The first centers on the question of whether FMF-associated mutations in MEFV (the gene mutated in FMF) have undergone natural selection in recent history, while the second focuses on the interactions between pyrin, the protein encoded by MEFV, and Yersinia pestis, the agent of the bubonic plague. Even before the identification of MEFV by positional cloning, population genetic studies indicated a very high carrier frequency for FMF heterozygotes in the Mediterranean basin and Middle East. With the availability of genetic testing, we and others have documented a heterozygote carrier frequency of approximately 10% in multiple Middle Eastern populations, with different mutations predominating in different populations. These findings are suggestive of positive selection, but they could still be the result of genetic drift. In order to test the hypothesis of selection, we performed haplotype analyses on 2,313 Turkish individuals who had undergone dense genome-wide single nucleotide polymorphism (SNP) genotyping. In 4,626 Turkish haplotypes including 91 with the MEFV_p.V726A mutation and 145 with the MEFV_p.M694V mutation, we found extended haplotype homozygosity among haplotypes bearing the mutations compared with haplotypes with the ancestral alleles. This extended homozygosity was greater than expected by two haplotype diversity tests, iHS and nSL, strongly suggesting recent positive selection. It is noteworthy that there was no evidence for positive selection of 232 haplotypes with MEFV_p.E148Q, a variant generally thought to be a functional polymorphism. Further analyses indicated that carriers of each mutation (p.V726A and p.M694V) are descended from common ancestors living at least 2,500 - 3,000 years ago. Given the evidence in favor of positive selection for FMF heterozygotes, we considered various microbial pathogens that might have selected for FMF-associated MEFV mutations. Previous studies from our laboratory demonstrated that inactivation of RhoA GTPase triggers a biochemical pathway leading to the assembly of the pyrin inflammasome, which activates the proinflammatory cytokines IL-1beta and IL-18. We have further shown that the Yersinia pestis YopE and YopT toxins inactivate RhoA and thereby stimulate pyrin inflammasome assembly, while the YopM Yersinia toxin blocks pyrin inflammasome activation by stimulating pyrin phosphorylation at residues 208 and 242 with subsequent binding of 14-3-3 inhibitory proteins to pyrin. In the present set of experiments, we explored the mechanism whereby YopM induces pyrin phosphorylation, and examined differences between wild type pyrin and FMF-associated mutants in their susceptibility to YopM-mediated phosphorylation in human peripheral blood leukocytes. We found that YopM not only induces pyrin phosphorylation by PKN1 and PKN2 (which we had shown previously), but also promotes the interaction of pyrin with RSK (ribosomal S6 kinase). Pyrin is not ordinarily a substrate for RSK, but in the presence of YopM RSK strongly phosphorylates pyrin at residues 208 and 242, thereby inducing 14-3-3 binding and blocking the pyrin inflammasome. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than wild type human pyrin, attenuating YopM-induced IL-1beta suppression. Relative to healthy controls, peripheral blood mononuclear cells from patients with biallelic mutations and heterozygous carriers released heightened IL-1beta in response to Yersinia, but not to Yersinia pestis YopM deletion mutants, or to Burkholderia cenocepacia (which lacks YopM). Mefv M680I/M680I knockin mice infected with Yersinia pestis exhibited IL-1beta-dependent increased survival relative to wild-type knockin mice. The data from this project demonstrate selection for FMF in Mediterranean populations, associated with heightened resistance to Yersinia. A manuscript describing these findings has been submitted for publication. 2) Deficiency of adenosine deaminase 2 (DADA2) In the previous reporting period, we demonstrated the efficacy of tumor necrosis factor (TNF) inhibitors in preventing strokes in patients with DADA2. With continued follow up of the initial cohort of 15 DADA2 patients who had 55 strokes in a total of 2077 patient-months before anti-TNF initiation, there have been no strokes in 733 patient-months of observation since the initiation of TNF inhibitors. Before anti-TNF treatment, 10 of the 15 patients were receiving glucocorticoids at an average dose of 0.53 mg per kilogram body weight per day prednisone equivalent, while at the time of data analysis all patients had been weaned off glucocorticoids. Before treatment, 10 patients had elevated acute-phase reactants, values that normalized after anti-TNF initiation. Of the 12 patients who had undergone elastography to quantify liver fibrosis, all had levels of less than 7 kPa (the cutoff value indicating no increase in hepatic stiffness associated with underlying liver disease) after anti-TNF therapy, including 6 who had levels of more than 7 kPa before therapy. There were no serious adverse events associated with anti-TNF therapy. A manuscript describing these results was published during the current reporting period in the New England Journal of Medicine. In collaboration with Peter Merkel and the Vasculitis Clinical Research Consortium, we have screened 117 patients with idiopathic PAN, all of whom tested negative for hepatitis B virus infection, for ADA2 mutations. Four (3.4%) had biallelic rare missense variants in ADA2 with a minor allele frequency of less than 0.005. Of the 7 distinct variants present in these 4 patients, 6 had previously been reported as causative for DADA2, and the remaining variant is computationally predicted to be damaging to protein function. Four additional patients were carriers for monoallelic variants, one of which has been reported in DADA2 before. Of 1107 patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA), none were biallelic for ADA2 mutations. This work was presented as a plenary abstract at the 2018 annual meeting of the American College of Rheumatology, and a manuscript has been submitted for publication. We have continued to accept referrals of patients with possible DADA2, and now have a cohort of over 50 patients. During the current reporting period we have identified 7 novel mutations: p.R34W, p.W204C, p.E244A, p.D329N, p.P425A, p.P435A, and p.W501*. Immunologic analyses demonstrated the overproduction of cytokines mediated by type I interferon and NF-kappaB pathways in patients' primary cells. Treatment with TNF inhibitors led to reduced inflammation, rescued the skewed differentiation towards the pro-inflammatory M1 macrophage subset, and restored the integrity of endothelial cells in blood vessels. A manuscript summarizing these findings will be submitted during the next reporting period.
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