A human immune dysregulation syndrome characterized by severe hyperinflammation with a homozygous nonsense Roquin-1 mutation

Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability
Flow Cytometry data was analyzed using FlowJo software (BD-Biosciences). Immunoblots were analyzed using Image Lab 5.2.1 Software (Bio-Rad). A voxel-based measurement of the tresholded PCC and Manders coefficients M1 en M2 was carried out in Volocity 6.3.0 (Perkin Elmer). Gene expression was analyzed using qbase+ software version 2.6 (Biogazelle). FlowSOM analysis was performed in R.
The data that support the findings of this study are available from the corresponding author upon request. The flowcytometry dataset used for FlowSOM analysis has been deposited in the public database FlowRepository as experiment FR-FCM-Z267.

nature research | reporting summary
October 2018 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. In case of studies performed on human material, sample size was dependent on the number of patients available for this study. No statistical method was used to predetermine sample size of mouse experiments. Instead sample size was determined based on the numbers required to generate statistical power and preliminary experiments.
No data exclusion was performed for this study.
All experiments were repeated at least twice. Data presented in this study is representative for the different repeats OR combined in one final figure.
In the case of human studies, patients samples were compared to healthy controls of similar age. In the case of murine studies, both mutant and wild type mice were randomized over housing cages. Mutant mice were compared to age and sex matched controls.
No blinding was performed for these studies as it was deemed as not relevant. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided. HEK 293T cells were not authenticated.
HEK293T were tested negative for Mycoplasma contamination prior to use in experiments.
No cell lines that have been reported as misidentified have been used in this study.
Sanroque mice carrying the M199R mutation in the ROQ domain of Rc3h1 were backcrossed to the C57Bl/6 background. C57Bl/6 CD45.1/CD45.2 mice were used to generate bone marrow chimeras. Rc3h1-2fl/fl; CD4-Cre-ERT2 mice were crossed with rtTA transgenic mice to generate C57Bl/6 Rc3h1-2fl/fl; CD4-Cre-ERT2; rtTA mice. Both male and female mice were used in the experiments. In the case of bone marrow transplantation, mice were tested 8 weeks post irradiation. Details are reported in Methods.
The study did not involve wild animals.
The study did not involve samples collected from the field.
All experimental procedures involving mice were performed in accordance with the regulations of and were approved by the Ludwig-Maximilians-Universität Mu" nchen or the ethical committee of the Science Department at Ugent University.
The patient studied in this manuscript is an 18yr old male of caucasian origin. A nonsense mutation (R688*) in the RC3H1 gene was identified by WES.
Patient recruitment is clinician guided on a case by case basis. No criteria of inclusion or exclusion were formulated for this study.
This study was approved by the ethical committee of Ghent University Hospital