Abstract
Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV, which encodes pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turkish people, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than with wild-type human pyrin, thereby attenuating YopM-induced interleukin (IL)-1β suppression. Relative to healthy controls, leukocytes from patients with FMF harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1β specifically in response to Y. pestis. Y. pestis-infected MefvM680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis.
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Data availability
The datasets generated and/or analyzed during the current study are available from the Supplementary Information and Source Data files in the online version of the paper and also available from the corresponding author on request.
Code availability
Computer code for the forward-time simulations is available at https://github.com/dshriner/forward-time-simulators under the GNU General Public License v.3.0.
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Acknowledgements
We thank the patients enrolled in our clinical protocols for providing research specimens, A. Jones, T. Romeo, L. Poe and R. Laird for help with caring for patients and A. Schaffer for thoughtful discussions. This work was supported by the Intramural Research Programs of the National Human Genome Research Institute, the National Institute of Allergy and Infectious Diseases, the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the Center for Research on Genomics and Global Health. This research was also supported by the National Institute of Allergy and Infectious Diseases award R01AI099222 (to J.B.B.) and utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov).
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J.J.C. conceived the project. Y.H.P., E.F.R., J.B.B., D.L.K. D.S. and J.J.C. designed the study. Y.H.P., E.F.R., W.L., L.K.C., M.I.I., N.A.L., Y.Z.A.-U., B.B.-P., D.S. and J.J.C. performed experiments. Y.H.P., E.F.R., W.L., Z.S., I.A., C.N.R., H.C., J.B.B., D.L.K., D.S. and J.J.C. analyzed the data. Y.H.P., E.F.R., I.A., C.N.R., J.B.B., D.L.K., D.S. and J.J.C. wrote the manuscript. A.K.O., D.L.S., K.S.B., P.H., M.N., E.S., I.A., A.G. and S.O. provided the clinical data and specimens for healthy controls, carriers and patients. B.B.-P. and S.Z. provided laboratory resources and assistance at the Hacettepe University Faculty of Medicine.
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Extended data
Extended Data Fig. 1 Haplotypes from 2,313 Turkish individuals provide evidence of evolutionarily recent positive selection and estimates of selection intensity and duration of selection on FMF-associated pyrin mutations.
a-c, Histograms of unstandardized nSL statistics of GWAS variants37 with similar frequency and local recombination rate as each FMF mutation. a, 1,402 markers similar to MEFV_p.V726A; b, 2,380 markers similar to MEFV_p.M694V; and c, 5,442 markers similar to MEFV_p.E148Q. The most extreme negative nSL values have the strongest evidence of recent positive selection. d-f, Log-likelihood plots and selection co-efficient estimates with 95% confidence intervals and mutation age estimates with 95% confidence intervals of three MEFV mutations, determined with a hidden Markov model method from the multi-locus haplotype structure of 4,626 Turkish chromosomes. d, MEFV_p.V726A; e, MEFV_p.M694V; and f, MEFV_p.E148Q. g-i, Histograms of iHH values for the ancestral alleles of markers with similar allele frequency and local recombination rate as each MEFV mutation, demonstrating that the ancestral alleles have not been under positive selection, thereby providing evidence that the mutations have not been under balancing selection. g, 1,570 markers similar to MEFV_ p.V726, h, 2,677 markers similar to MEFV_p.M694, and i, 6,166 markers similar to MEFV_p.E148.
Extended Data Fig. 2 Geographically diverse FMF mutation carriers exhibit extended conserved haplotypes.
a, MEFV_p.M694V conserved haplotypes (yellow). The length of the MEFV_p.M694V haplotype shared by all the diverse carrier chromosomes = 48 kb and shared by greater than 50% of the diverse carrier chromosomes = 307 kb. b, MEFV_p.V726A conserved haplotypes (pink). The length of the MEFV_p.V726A haplotype shared by all the diverse carrier chromosomes = 77 kb and shared by greater than 50% of the diverse carrier chromosomes = 262 kb. The fully conserved shared haplotypes in the diverse mutation carriers are identical to the respective haplotypes in the Turkish population.
Extended Data Fig. 3 Y. pestis YopM suppresses the pyrin inflammasome.
a,b, IL-1β measurements of culture supernatants of Mefv+/+ BMDMs primed with LPS, treated with or without C3 toxin, and infected with indicated MOI of WT (a) or ∆yopM (b) Y. pestis strains. Results are presented as mean ± s.e.m., for n = 5 independent biological replicates. c, Immunoblot analysis of pyrin and pro-IL-1β in lysates of retroviral transduced U937 cells, expressing WT or indicated Ser to Ala mutant pyrin proteins. Data are representative of three independent experiments with similar results.
Extended Data Fig. 4 RSK-YopM mediated pyrin phosphorylation is independent of PKN.
In vitro kinase assay of purified Myc/His-tagged N-terminal human pyrin (amino acids 1–330) incubated with recombinant PKN1 and/or RSK1 (a) or PKN2 and/or RSK1 (b) in the presence of purified GST or GST-YopM, and analyzed by immunoblot with antibody specific for S242 phosphorylated pyrin (a) or phosphorylated serine (b). Data are representative of three independent experiments with similar results.
Extended Data Fig. 5 5 Human and murine RSK isoforms exhibit a restricted distribution of gene expression in leukocytes.
a,b, Relative gene expression profile of RSK isoforms in human PBMCs with or without Y. pestis infection (a) and mouse BMDMs with or without LPS priming (b), and assayed by RT-QPCR. Results are presented as mean ± s.e.m., for n = 5 independent biological replicates.
Extended Data Fig. 6 Knockdown of RSK1, RSK2 and RSK3 in THP-1 cells induces ASC oligomerization.
a, Immunoblot analysis of lysates of THP-1 cells transiently transfected with negative control siRNA with no substantial sequence similarity to human gene sequences (N.C.) or a mixture of siRNAs targeting RSK1, RSK2 and RSK3. b, ASC oligomerization analysis by immunoblot from THP-1 cells transfected with N.C. or a mixture of siRNAs targeting RSK1, RSK2 and RSK3, and infected with Y. pestis (MOI 30). Cell lysates and the disuccinimidyl suberate (DSS)-treated pellets were analyzed by immunoblot with anti-ASC antibody. Data are representative of three independent experiments with similar results.
Extended Data Fig. 7 YopM binds to multiple regions of N-terminal pyrin.
a, Schematic structure of human pyrin with various deletion fragments of N-terminal human pyrin. b,c, GST-pulldown assay of V5-tagged various N-terminal human pyrin fragments with purified GST-YopM. Data are representative of three independent experiments with similar results.
Supplementary information
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Park, Y.H., Remmers, E.F., Lee, W. et al. Ancient familial Mediterranean fever mutations in human pyrin and resistance to Yersinia pestis. Nat Immunol 21, 857–867 (2020). https://doi.org/10.1038/s41590-020-0705-6
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DOI: https://doi.org/10.1038/s41590-020-0705-6
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